Analogs of benzoquinone-containing ansamycins and methods of use thereof

ABSTRACT

The present invention provides analogs of benzoquinone-containing ansamycins and uses thereof for treating and modulating disorders associated with hyperproliferation, such as cancer. The present invention provides analogs of benzoquinone-containing ansamycins where the benzoquinone is reduced to a hydroquinone and trapped by reaction with a suitable acid, preferably ones that increase the solubility and air stability of the resulting 17-ammonium hydroquinone ansamycin analog.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/199,578, filed Aug. 27, 2008, which is a continuation of U.S. patentapplication Ser. No. 11/105,203, filed Apr. 13, 2005, now U.S. Pat. No.7,566,706, which is a continuation of U.S. patent application Ser. No.11/022,057, filed Dec. 23, 2004, now U.S. Pat. No. 7,282,493, whichclaims the benefit of priority to U.S. Provisional Patent ApplicationSer. No. 60/532,080, filed Dec. 23, 2003; U.S. Provisional PatentApplication Ser. No. 60/540,142, filed Jan. 29, 2004; U.S. ProvisionalPatent Application Ser. No. 60/547,381, filed Feb. 23, 2004; U.S.Provisional Patent Application Ser. No. 60/561,718, filed Apr. 12, 2004;U.S. Provisional Patent Application Ser. No. 60/567,565, filed May 3,2004; U.S. Provisional Patent Application Ser. No. 60/606,283, filedSep. 1, 2004; U.S. Provisional Patent Application Ser. No. 60/626,286,filed Nov. 9, 2004; U.S. Provisional Patent Application Ser. No.60/632,858, filed Dec. 3, 2004; the specifications of all of them arehereby incorporated in their entirety.

BACKGROUND OF THE INVENTION

Geldanamycin is a macrocyclic lactam that is a member of thebenzoquinone-containing ansamycins family of natural products. Theisolation, preparation and various uses of geldanamycin are described inU.S. Pat. No. 3,595,955. Like most naturally-occurring members of thisclass of molecules, geldanamycin is typically produced as a fermentationproduct of Streptomyces hygroscopicus var. geldanus var. nova strain(Journal of Antibiotics Vol. 23, Page 442 (1970)). Other analogs andderivatives of geldanamycin have been identified or synthesized, andtheir use as antitumor agents is described in U.S. Pat. Nos. 4,261,989and 5,387,584, and published PCT applications WO 00/03737 and WO03/072794. One member of this family that has been examined in somedetail is 17-allylamino-17-demethoxygeldanamycin (“17-AAG”).Geldanamycin and its derivative have been shown to bind to HSP90 andantagonize the protein's activity.

HSP90 is a highly abundant protein which is essential for cell viabilityand it exhibits dual chaperone functions (J. Cell Biol. (2001)154:267-273, Trends Biochem. Sci. (1999) 24:136-141). It plays a keyrole in the cellular stress response by interacting with many proteinsafter their native conformation has been altered by variousenvironmental stresses, such as heat shock, ensuring adequate proteinfolding and preventing non-specific aggregation (Pharmacological Rev.(1998) 50:493-513). In addition, recent results suggest that HSP90 mayalso play a role in buffering against the effects of mutation,presumably by correcting the inappropriate folding of mutant proteins(Nature (1998) 396:336-342). However, HSP90 also has an importantregulatory role under normal physiological conditions and is responsiblefor the conformational stability and maturation of a number of specificclient proteins, of which about 40 are known (see. Expert. Opin. BiolTher. (2002) 2(1): 3-24). These can be subdivided into three groups:steroid hormone receptors, serine/threonine or tyrosine kinases and acollection of apparently unrelated proteins, including mutant p53 andthe catalytic subunit of telomerase hTERT. All of these proteins playregulatory roles in physiological and biochemical processes in the cell.

HSP90 antagonists are currently being explored in a large number ofbiological contexts where a therapeutic effect can be obtained for acondition or disorder by inhibiting one or more aspects of HSP90activity. Although the primary focus has been on proliferativedisorders, such as cancers, other conditions are showing levels oftreatment using HSP90 antagonist. For example, U.S. Published PatentApplication 2003/0216369, discloses the use of HSP90 inhibitors fortreatment of viral disorders. HSP90 inhibitors have also been implicatedin a wide variety of other utilities, including use as anti-inflammationagents, agents for treating autoimmunity, agents for treating stroke,ischemia, cardiac disorders and agents useful in promoting nerveregeneration (See, e.g., WO 02/09696 (PCT/US01/23640); WO 99/51223(PCT/US99/07242); U.S. Pat. No. 6,210,974 B1; and U.S. Pat. No.6,174,875). There are reports in the literature that fibrogeneticdisorders including but not limited to scleroderma, polymyositis,systemic lupus, rheumatoid arthritis, liver cirrhosis, keloid formation,interstitial nephritis, and pulmonary fibrosis may be treatable usingHSP90 inhibitors. (Strehlow, WO 02/02123; PCT/US01/20578).

Geldanamycin's nanomolar potency and apparent selectivity for killingtumor cells, as well as the discovery that its primary target inmammalian cells is HSP90, has stimulated interest in its development asan anti-cancer drug. However, the extremely low solubility of thesemolecules and the association of hepatotoxicity with the administrationof geldanamycin has led to difficulties in developing an approvableagent for therapeutic applications. In particular, geldanamycin has poorwater solubility, making it difficult to deliver in therapeuticallyeffective doses.

More recently, attention has focused on 17-amino derivatives ofgeldanamycin, in particular 17-AAG, that show reduced hepatotoxicitywhile maintaining HSP90 binding. See U.S. Pat. Nos. 4,261,989;5,387,584; and 5,932,566. Like geldanamycin, 17-AAG has very limitedaqueous solubility. This property requires the use of a solubilizingcarrier, e.g., egg phospholipid with DMSO, or Cremophore® (BASFAktiengesellschaft), a polyethoxylated castor oil; the presence ofeither of these carriers results in serious side reactions in somepatients.

Consequently, there remains a need to discover more soluble analogs ofbenzoquinone-containing ansamycins and specific and general methods forcreating them, particularly geldanamycin and its analogs, such as17-AAG.

SUMMARY OF THE INVENTION

The present invention provides reduced forms of benzoquinone-containingansamycins, and salts thereof in isolated form and in pharmaceuticalpreparations, and uses of them for treating and modulating disordersassociated with hyperproliferation, such as cancer. Generally, thepresent invention provides soluble, stable drug forms ofbenzoquinone-containing ansamycins. The present invention providesreduced analogs of benzoquinone-containing ansamycins, such as 17-aminoanalogs of geldanamycin in isolated form and in pharmaceuticalpreparations, wherein the benzoquinone is reduced to a hydroquinone andtrapped in an air-stable and isolated form, such as an HCl or H₂SO₄salt. Alternatively, the hydroquinones may be trapped as co-salts withan amino acid such as glycine. Such analogs are remarkably water soluble(1-3 orders of magnitude more soluble than the non-reduced form, e.g.,35 μg/mL for 17-AAG vs. 1-3 mg/mL for the hydroquinone of 17-AAG,and >200 mg/mL for salts of hydroquinone derivatives of 17-AAG) andstable; and they can be isolated and formulated for human administrationwithout the problems associated with the formulation, storage andinstability of the non-reduced parent forms and other formulations ofansamycins.

In one embodiment, the present invention provides a pure and isolatedcompound of formula 1:

or the free base thereof;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)₁₃]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(OR₁₉),and —P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 1 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In another embodiment the present invention provides a pure and isolatedcompound with absolute sterochemistry as shown in formula 2:

or the free base thereof;

wherein independently for each occurrence:

X⁻ is selected from the group consisting of chloride, bromide, iodide,H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate, p-toluenesulfonate,trifluoromethylsulfonate, 10-camphorsulfonate, naphthalene-1-sulfonicacid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, cyclamic acid salt,thiocyanic acid salt, naphthalene-2-sulfonate, and oxalate.

R₁ is hydroxyl or —OC(O)R₈;

R₃ and R₄ are hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl,heteroaralkyl, or —[(CR₂)_(p)]—R₁₈; or R₃ taken together with R₄represent a 4-8 membered optionally substituted heterocyclic ring;

R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₇)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(OR₁₉),and —P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₇ is hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, or heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₇ is hydrogen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the stereochemistry of a double bond may be E or Z or a mixture thereof.

In another embodiment the present invention provides a pure and isolatedcompound with absolute sterochemistry as shown in formula 3:

wherein X⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate.

In one embodiment the present invention provides a compound of formula4:

or a pharmaceutically acceptable salt thereof;

wherein, independently for each occurrence,

W is oxygen or sulfur;

Z is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

n is equal to 0, 1, or 2;

m is equal to 0, 1, or 2;

X and Y are independently C(R₃₀)₂; wherein R₃₀ for each occurrence isindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl; or—[(CR₂)_(p)]—R₁₆;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆;

R₄ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the Formula 4a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₅ and R₆ are both hydrogen; or R₅ and R₆ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(OR₁₉),and —P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; and

the absolute stereochemistry at a stereogenic center of formula 4 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In another embodiment the present invention provides a compound withabsolute sterochemistry as shown in formula 5:

wherein independently for each occurrence:

n is equal to 0, 1, or 2;

m is equal to 0, 1, or 2;

X and Y are independently C(R₃₀)₂; wherein R₃₀ for each occurrence isindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl; or—[(CR₂)_(p)]—R₁₆;

R₁ is hydroxyl or —OC(O)R₈;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl,heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₅ and R₆ are both hydrogen; or R₅ and R₆ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₇ is hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, or heteroaralkyl; and

the stereochemistry of a double bond may be E or Z or a mixture thereof.

In another embodiment the present invention provides a compound selectedfrom the group consisting of:

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising: at least one pharmaceutically acceptableexcipient; and a compound of formula 6:

or the free base thereof;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆, or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 6a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 6 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising: at least one pharmaceutically acceptableexcipient; a compound of formula 6:

or the free base thereof; and a compound of formula 10, wherein saidcompound of formula 10 is present in the range of about 0.00001% toabout 5% (m/v):

or pharmaceutically acceptable salt thereof;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 6a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 6 or 10may be R or S or a mixture thereof and the stereochemistry of a doublebond may be E or Z or a mixture thereof.

In other embodiments, the present invention relates to a method oftreating cancer, comprising administering to a mammal in need thereof atherapeutically effective amount of a compound of the present invention;or a therapeutically effective amount of a pharmaceutical composition ofthe present invention.

Another aspect of the invention relates to a method of preparing acompound, comprising: combining a compound of formula 7 with a reducingagent in a reaction solvent to give a compound of formula 8; and

combining said compound of formula 8 with a pharmaceutically acceptableacid to give said compound of formula 1;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 1 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts chromatograms from a LCMS analysis of the DimethylaminoAcetate Co-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 3.

FIG. 2 depicts mass spectra from a LCMS analysis of the DimethylaminoAcetate Co-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 3.

FIG. 3 depicts a ¹H NMR spectrum of the α-Aminoisobutyrate Co-Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 4.

FIG. 4 depicts chromatograms from a LCMS analysis of theα-Aminoisobutyrate Co-Salt of the Hydroquinone of 17-AAG preparedaccording to the procedure described in Example 4.

FIG. 5 depicts a mass spectrum from a LCMS analysis of theα-Aminoisobutyrate Co-Salt of the Hydroquinone of 17-AAG preparedaccording to the procedure described in Example 4.

FIG. 6 depicts a ¹H NMR spectrum of the β-Alanine Co-Salt of theHydroquinone of 17-AAG prepared according to the procedure described inExample 5.

FIG. 7 depicts chromatograms from a LCMS analysis of the β-AlanineCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 5.

FIG. 8 depicts mass spectra from a LCMS analysis of the β-AlanineCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 5.

FIG. 9 depicts a ¹H NMR spectrum of the N-Methyl Glycine Co-Salt of theHydroquinone of 17-AAG prepared according to the procedure described inExample 6.

FIG. 10 depicts chromatograms from a LCMS analysis of the N-MethylGlycine Co-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 6.

FIG. 11 depicts mass spectra from a LCMS analysis of the N-MethylGlycine Co-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 6.

FIG. 12 depicts a ¹H NMR spectrum of the Piperidine Carboxylate Co-Saltof the Hydroquinone of 17-AAG prepared according to the proceduredescribed in Example 7.

FIG. 13 depicts chromatograms from a LCMS analysis of the PiperidineCarboxylate Co-Salt of the Hydroquinone of 17-AAG prepared according tothe procedure described in Example 7.

FIG. 14 depicts mass spectra from a LCMS analysis of the PiperidineCarboxylate Co-Salt of the Hydroquinone of 17-AAG prepared according tothe procedure described in Example 7.

FIG. 15 depicts mass spectra from a LCMS analysis of the PiperidineCarboxylate Co-Salt of the Hydroquinone of 17-AAG prepared according tothe procedure described in Example 7.

FIG. 16 depicts a ¹H NMR spectrum of the Glycine Co-Salt of theHydroquinone of 17-AAG prepared according to the procedure described inExample 8.

FIG. 17 depicts chromatograms from a LCMS analysis of the GlycineCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 8.

FIG. 18 depicts mass spectra from a LCMS analysis of the Glycine Co-Saltof the Hydroquinone of 17-AAG prepared according to the proceduredescribed in Example 8.

FIG. 19 depicts a ¹H NMR spectrum of the 2-Amino-2-ethyl-butyrateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 9.

FIG. 20 depicts chromatograms from a LCMS analysis of the2-Amino-2-ethyl-butyrate Co-Salt of the Hydroquinone of 17-AAG preparedaccording to the procedure described in Example 9.

FIG. 21 depicts mass spectra from a LCMS analysis of the2-Amino-2-ethyl-butyrate Co-Salt of the Hydroquinone of 17-AAG preparedaccording to the procedure described in Example 9.

FIG. 22 depicts a ¹H NMR spectrum of the 1-Amino-CyclopropanecarboxylateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 10.

FIG. 23 depicts chromatograms from a LCMS analysis of the1-Amino-Cyclopropanecarboxylate Co-Salt of the Hydroquinone of 17-AAGprepared according to the procedure described in Example 10.

FIG. 24 depicts mass spectra from a LCMS analysis of the1-Amino-Cyclopropanecarboxylate Co-Salt of the Hydroquinone of 17-AAGprepared according to the procedure described in Example 10.

FIG. 25 depicts a ¹H NMR spectrum of the Carboxylate Co-Salt of theHydroquinone of 17-AAG prepared according to the procedure described inExample 11.

FIG. 26 depicts chromatograms from a LCMS analysis of the CarboxylateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 11.

FIG. 27 depicts mass spectra from a LCMS analysis of the CarboxylateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 11.

FIG. 28 depicts a ¹H NMR spectrum of the 1-Amino-cyclopentanecarboxylateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 12.

FIG. 29 depicts chromatograms from a LCMS analysis of the1-Amino-cyclopentanecarboxylate Co-Salt of the Hydroquinone of 17-AAGprepared according to the procedure described in Example 12.

FIG. 30 depicts mass spectra from a LCMS analysis of the1-Amino-cyclopentanecarboxylate Co-Salt of the Hydroquinone of 17-AAGprepared according to the procedure described in Example 12.

FIG. 31 depicts a ¹H NMR spectrum of the N-Methyl PiperidinecarboxylateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 13.

FIG. 32 depicts chromatograms from a LCMS analysis of the N-MethylPiperidinecarboxylate Co-Salt of the Hydroquinone of 17-AAG preparedaccording to the procedure described in Example 13.

FIG. 33 depicts mass spectra from a LCMS analysis of the N-MethylPiperidinecarboxylate Co-Salt of the Hydroquinone of 17-AAG preparedaccording to the procedure described in Example 13.

FIG. 34 depicts a ¹H NMR spectrum of the N,N,N-Trimethylammonium AcetateCo-Salt of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 14.

FIG. 35 depicts a ¹H NMR spectrum of the Air-stable HydroquinoneDerivatives of 17-AAG Geldanamycin prepared according to the proceduredescribed in Example 15.

FIG. 36 depicts a ¹H NMR spectrum of the HCl salt of the HydroquinoneDerivative of 17-AAG Geldanamycin prepared according to the proceduredescribed in Example 17.

FIG. 37 depicts chromatograms from a LCMS analysis of the HCl Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 17.

FIG. 38 depicts a ¹H NMR spectrum of the H₂SO₄ salt of the HydroquinoneDerivative of 17-AAG Geldanamycin prepared according to the proceduredescribed in Example 18.

FIG. 39 depicts chromatograms from a LCMS analysis of the H₂SO₄ Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 18.

FIG. 40 depicts mass spectra from a LCMS analysis of the H₂SO₄ Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 18.

FIG. 41 depicts chromatograms from a LCMS analysis of thep-Toluenesulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 19.

FIG. 42 depicts mass spectra from a LCMS analysis of thep-Toluenesulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 19.

FIG. 43 depicts mass spectra from a LCMS analysis of thep-Toluenesulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 19.

FIG. 44 depicts chromatograms from a LCMS analysis of thed-Camphorsulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 20.

FIG. 45 depicts mass spectra from a LCMS analysis of thed-Camphorsulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 20.

FIG. 46 depicts mass spectra from a LCMS analysis of thed-Camphorsulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 20.

FIG. 47 depicts mass spectra from a LCMS analysis of thed-Camphorsulfonic Salt of the Hydroquinone of 17-AAG prepared accordingto the procedure described in Example 20.

FIG. 48 depicts chromatograms from a LCMS analysis of the H₃PO₄ Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 21.

FIG. 49 depicts mass spectra from a LCMS analysis of the H₃PO₄ Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 21.

FIG. 50 depicts chromatograms from a LCMS analysis of the MeSO₃H Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 22.

FIG. 51 depicts mass spectra from a LCMS analysis of the MeSO₃H Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 22.

FIG. 52 depicts chromatograms from a LCMS analysis of the PhSO₃H Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 23.

FIG. 53 depicts mass spectra from a LCMS analysis of the PhSO₃H Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 23.

FIG. 54 depicts a ¹H NMR spectrum of the cyclic carbamate of theHydroquinone of 17-AAG prepared according to the procedure described inExample 25.

FIG. 55 depicts chromatograms from a LCMS analysis of the cycliccarbamate of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 25.

FIG. 56 depicts a mass spectrum from a LCMS analysis of the cycliccarbamate of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 25.

FIG. 57 depicts a ¹H NMR spectrum of the lactam of the Hydroquinone of17-AAG prepared according to the procedure described in Example 26.

FIG. 58 depicts chromatograms from a LCMS analysis of the lactam of theHydroquinone of 17-AAG prepared according to the procedure described inExample 26.

FIG. 59 depicts a mass spectrum from a LCMS analysis of the lactam ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 26.

FIG. 60 depicts a ¹H NMR spectrum of a 17-amino derivative of theHydroquinone of 17-AAG prepared according to the procedure described inExample 27.

FIG. 61 depicts chromatograms from a LCMS analysis of a 17-aminoderivative of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 27.

FIG. 62 depicts a mass spectrum from a LCMS analysis of a 17-aminoderivative of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 27.

FIG. 63 depicts a ¹H NMR spectrum of a 17-(3-amino-propane-1,2-diol)derivative of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 28.

FIG. 64 depicts chromatograms from a LCMS analysis of a17-(3-amino-propane-1,2-diol) derivative of the Hydroquinone of 17-AAGprepared according to the procedure described in Example 28.

FIG. 65 depicts a mass spectrum from a LCMS analysis of a17-(3-amino-propane-1,2-diol) derivative of the Hydroquinone of 17-AAGprepared according to the procedure described in Example 28.

FIG. 66 depicts chromatograms from a LCMS analysis of a BODIPYderivative of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 29.

FIG. 67 depicts a mass spectrum from a LCMS analysis of a BODIPYderivative of the Hydroquinone of 17-AAG prepared according to theprocedure described in Example 29.

FIG. 68 depicts a ¹H NMR spectrum of the HBr Salt of the Hydroquinone of17-AAG prepared according to the procedure described in Example 31.

FIG. 69 depicts chromatograms from a LCMS analysis of the HBr Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 31.

FIG. 70 depicts a mass spectrum from a LCMS analysis of the HBr Salt ofthe Hydroquinone of 17-AAG prepared according to the procedure describedin Example 31.

DETAILED DESCRIPTION OF THE INVENTION

Overview

The present invention provides pure and isolated, reduced forms ofanalogs of benzoquinone-containing ansamycins, salts and intermediatesthereto. The present invention also provides methods for the use ofthese compounds in the treatment of diseases or conditions characterizedby undesired cellular hyperproliferation, such as cancers, as well asother conditions and disorders associated with unwanted HSP90 activityor in which HSP90 plays a role in the cells involved in causing thedisorder. The present invention provides reduced analogs ofbenzoquinone-containing ansamycins where the benzoquinone is reduced toa hydroquinone, and preferably isolated and purified as a salt form. Inan alternative embodiment a compound of the present invention isco-crystallized with an amino acid salt. Such analogs, either with orwithout the amino acid salt, are remarkably water soluble (1-3 orders ofmagnitude greater solubility than the non-reduced form, e.g., 35 μg/mL17-AAG vs. 1-3 mg/mL for the hydroquinone of 17-AAG and >200 mg/mL forthe salt of the hydroquinone), and stable at room temperature; and theycan be isolated and formulated for human administration without theproblems associated with the formulation, storage and instability of thenon-reduced parent forms and other formulations of ansamycins.

DEFINITIONS

The definitions of terms used herein are meant to incorporate thepresent state-of-the-art definitions recognized for each term in thechemical and pharmaceutical fields. Where appropriate, exemplificationis provided. The definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

Where stereochemistry is not specifically indicated, all stereoisomersof the inventive compounds are included within the scope of theinvention, as pure compounds as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythe present invention. Polymorphic crystalline forms and solvates arealso encompassed within the scope of this invention.

As used herein, the term “amino acid” refers to molecules containingboth a carboxylic acid moiety and an amino moiety. The carboxylic acidand amino moeities are as defined below. Both naturally occurring andsynthetically derived amino acids are encompassed in the scope of thisinvention.

As used herein, the term “benzoquinone ansamycin” refers to a compoundcomprising a macrocyclic lactam, further comprising only one lactam inthe ring and a benzoquinone moiety in the lactam ring, wherein saidbenzoquinone moiety has at least one nitrogen substituent, wherein oneof said at least one nitrogen substitutents is part of said only oneamide moiety in the lactam ring. Specific examples ofnaturally-occurring benzoquinone ansamycins that can be used in thepresent invention include, but are not limited to, geldanamycin andherbimycin. The term “geldanamycin analog” refers to a benzoquinoneansamycin that can be derived from geldanamycin e.g., by chemicalmanipulation; for example 17-allylamino-17-demethoxygeldanamycin(17-AAG) or 17-(2-dimethylaminoethy)amino-17-demethoxygeldanamycin(17-DMAG).

As used herein, the term “isolated” in connection with a compound of thepresent invention means the compound is not in a cell or organism andthe compound is separated from some or all of the components thattypically accompany it in nature.

As used herein, the term “pure” in connection with an isolated sample ofa compound of the present invention means the isolated sample containsat least 60% by weight of the compound. Preferably, the isolated samplecontains at least 70% by weight of the compound. More preferably, theisolated sample contains at least 80% by weight of the compound. Evenmore preferably, the isolated sample contains at least 90% by weight ofthe compound. Most preferably, the isolated sample contains at least 95%by weight of the compound. The purity of an isolated sample of acompound of the present invention may be assessed by a number of methodsor a combination of them; e.g., thin-layer, preparative or flashchromatography, mass spectrometry, HPLC, NMR analysis, and the like.

The term “heteroatom” is art-recognized and refers to an atom of anyelement other than carbon or hydrogen. Illustrative heteroatoms includeboron, nitrogen, oxygen, phosphorus, sulfur and selenium.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In certain embodiments,a straight chain or branched chain alkyl has about 30 or fewer carbonatoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ forbranched chain), and alternatively, about 20 or fewer. Likewise,cycloalkyls have from about 3 to about 10 carbon atoms in their ringstructure, and alternatively about 5, 6 or 7 carbons in the ringstructure.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to aboutten carbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “aralkyl” is art-recognized and refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

The terms “alkenyl” and “alkynyl” are art-recognized and refer tounsaturated aliphatic groups analogous in length and possiblesubstitution to the alkyls described above, but that contain at leastone double or triple bond respectively.

The term “aryl” is art-recognized and refers to 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, naphthalene, anthracene, pyrene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics.” The aromaticring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms “heterocyclyl”, “heteroaryl”, or “heterocyclic group” areart-recognized and refer to 3- to about 10-membered ring structures,alternatively 3- to about 7-membered rings, whose ring structuresinclude one to four heteroatoms. Heterocycles may also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “optionally substituted” refers to a chemical group, such asalkyl, cycloalkyl aryl, and the like, wherein one or more hydrogen maybe replaced with a with a substituent as described herein, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like

The terms “polycyclyl” or “polycyclic group” are art-recognized andrefer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbonsare common to two adjoining rings, e.g., the rings are “fused rings”.Rings that are joined through non-adjacent atoms are termed “bridged”rings. Each of the rings of the polycycle may be substituted with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” is art-recognized and refers to —SO₂⁻. “Halide” designates the corresponding anion of the halogens, and“pseudohalide” has the definition set forth on 560 of “AdvancedInorganic Chemistry” by Cotton and Wilkinson.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R61 represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In other embodiments, R50 and R51(and optionally R52) each independently represent a hydrogen, an alkyl,an alkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carboxyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 andR56 represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61 or apharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R61, where m and R61 are defined above. WhereX50 is an oxygen and R55 or R56 is not hydrogen, the formula representsan “ester”. Where X50 is an oxygen, and R55 is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR55 is a hydrogen, the formula represents a “carboxylic acid”. Where X50is an oxygen, and R56 is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X50 is asulfur and R55 or R56 is not hydrogen, the formula represents a“thiolester.” Where X50 is a sulfur and R55 is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X50 is a sulfur and R56 ishydrogen, the formula represents a “thiolformate.” On the other hand,where X50 is a bond, and R55 is not hydrogen, the above formularepresents a “ketone” group. Where X50 is a bond, and R55 is hydrogen,the above formula represents an “aldehyde” group.

The term “carbamoyl” refers to —O(C═O)NRR′, where R and R′ areindependently H, aliphatic groups, aryl groups or heteroaryl groups.

The term “oxo” refers to a carbonyl oxygen (═O).

The terms “oxime” and “oxime ether” are art-recognized and refer tomoieties that may be represented by the general formula:

wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61. The moiety is an “oxime” when R is H; and itis an “oxime ether” when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl,aralkyl, or —(CH₂)_(m)—R61.

The terms “alkoxyl” or “alkoxy” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as may berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl,—O—(CH₂)_(m)—R61, where m and R61 are described above.

The term “sulfonate” is art recognized and refers to a moiety that maybe represented by the general formula:

in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R57 is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R50 and R56 are as defined above.

The term “sulfamoyl” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R50 and R51 are as defined above.

The term “sulfonyl” is art-recognized and refers to a moiety that may berepresented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” is art-recognized and refers to a moiety that maybe represented by the general formula:

in which R58 is defined above.

The term “phosphoryl” is art-recognized and may in general berepresented by the formula:

wherein Q50 represents S or O, and R59 represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl may be represented by thegeneral formulas:

wherein Q50 and R59, each independently, are defined above, and Q51represents O, S or N. When Q50 is S, the phosphoryl moiety is a“phosphorothioate”.

The term “phosphoramidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above.

The term “phosphonamidite” is art-recognized and may be represented inthe general formulas:

wherein Q51, R50, R51 and R59 are as defined above, and R60 represents alower alkyl or an aryl.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, and the like, whenit occurs more than once in any structure, is intended to be independentof its definition elsewhere in the same structure.

The term “selenoalkyl” is art-recognized and refers to an alkyl grouphaving a substituted seleno group attached thereto. Exemplary“selenoethers” which may be substituted on the alkyl are selected fromone of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R61, m andR61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention mayexist in particular geometric or stereoisomeric forms. In addition,polymers of the present invention may also be optically active. Thepresent invention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991). Protected forms of the inventive compounds are included withinthe scope of this invention.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term “pharmaceutically acceptable salt” or “salt” refers to a saltof one or more compounds. Suitable pharmaceutically acceptable salts ofcompounds include acid addition salts which may, for example, be formedby mixing a solution of the compound with a solution of apharmaceutically acceptable acid, such as hydrochloric acid, hydrobromicacid, sulfuric acid, fumaric acid, maleic acid, succinic acid, benzoicacid, acetic acid, citric acid, tartaric acid, phosphoric acid, carbonicacid, or the like. Where the compounds carry one or more acidicmoieties, pharmaceutically acceptable salts may be formed by treatmentof a solution of the compound with a solution of a pharmaceuticallyacceptable base, such as lithium hydroxide, sodium hydroxide, potassiumhydroxide, tetraalkylammonium hydroxide, lithium carbonate, sodiumcarbonate, potassium carbonate, ammonia, alkylamines, or the like.

The term “pharmaceutically acceptable acid” refers to inorganic ororganic acids that exhibit no substantial toxicity. Examples ofpharmaceutically acceptable acids include, but are not limited tohydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phenylsulfonic acid, methanesulfonic acid, fumaric acid, maleic acid,succinic acid, benzoic acid, acetic acid, citric acid, tartaric acid,phosphoric acid, carbonic acid, and the like.

The term “co-salt” or “co-crystal” refers to compositions in which thereduced salt form of the ansamycin is present with at least one othersalt, such as a salt of an amino acid.

The term “subject” as used herein, refers to an animal, typically amammal or a human, that will be or has been the object of treatment,observation, and/or experiment. When the term is used in conjunctionwith administration of a compound or drug, then the subject has been theobject of treatment, observation, and/or administration of the compoundor drug.

The terms “co-administration” and “co-administering” refer to bothconcurrent administration (administration of two or more therapeuticagents at the same time) and time varied administration (administrationof one or more therapeutic agents at a time different from that of theadministration of an additional therapeutic agent or agents), as long asthe therapeutic agents are present in the patient to some extent at thesame time.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits abiological or medicinal response in a cell culture, tissue system,animal, or human that is being sought by a researcher, veterinarian,clinician, or physician, which includes alleviation of the symptoms ofthe disease, condition, or disorder being treated.

The term “composition” is intended to encompass a product comprising thespecified ingredients in the specified amounts, as well as any productthat results, directly or indirectly, from combinations of the specifiedingredients in the specified amounts, particularly co-salts such as thereduced ansamycin salt (e.g., sulfate) with a salt of an amino acid(e.g., glycine).

The term “HSP90 mediated disorder” or “disorder mediated by cellsexpressing HSP90” refers to pathological and disease conditions in whichHSP90 plays a role. Such roles can be directly related to thepathological condition or can be indirectly related to the condition.The common feature to this class of conditions is that the condition canbe ameliorated by inhibiting the activity, function, or association withother proteins of HSP90.

The term “pharmaceutically acceptable carrier” refers to a medium thatis used to prepare a desired dosage form of a compound. Apharmaceutically acceptable carrier can include one or more solvents,diluents, or other liquid vehicles; dispersion or suspension aids;surface active agents; isotonic agents; thickening or emulsifyingagents; preservatives; solid binders; lubricants; and the like.Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1975) and Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe ed. (American PharmaceuticalAssoc. 2000), disclose various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present invention addresses the need to generate soluble forms ofansamycins, particularly members of the benzoquinone-containingfamilies, such as geldanamycin. Members of these classes of macrocyclicmolecules tend to be very insoluble, leading to poor profiles aspotential drugs (for example, 17-AAG has a solubility of only 100 μg/mLin an aqueous solution). The present invention solves these problems byproviding general reaction schemes that can be used to create analogs ofthese molecules that have improved solubility. The reaction schemesinclude reducing the quinone of such molecules to form a hydroquinone,and trapping it as a salt, such as HCl or H₂SO₄ salt. Remarkably, forexample, the hydroquinone HCl salt of 17-AAG has a solubility>about 200mg/mL.

Compounds

The present invention also provides the isolated analogs ofbenzoquinone-containing ansamycins, wherein the benzoquinone is reducedto a hydroquinone and trapped as the ammonium salt by reaction of thehydroquinone with a suitable organic or inorganic acid.

In one embodiment, the present invention provides a pure and isolatedcompound of formula 1:

or the free base thereof;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 1 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, provided thatwhen R₁ is hydroxyl, R₂ is hydrogen, R₅ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂₀, R₂₁,R₂₂, R₂₃, R₂₄, and R₂₅ are methyl; R₂₆ is hydrogen, Q is a bond; and Wis oxygen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein saidpharmaceutically acceptable acid has a pKa between about −10 and about 7in water.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein saidpharmaceutically acceptable acid has a pKa between about −10 and about 4in water.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein saidpharmaceutically acceptable acid has a pKa between about −10 and about 1in water.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein saidpharmaceutically acceptable acid has a pKa between about −10 and about−3 in water.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein X⁻ isselected from the group consisting of chloride, bromide, iodide, H₂PO₄⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate, p-toluenesulfonate,trifluoromethylsulfonate, 10-camphorsulfonate, naphthalene-1-sulfonicacid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, cyclamic acid salt,thiocyanic acid salt, naphthalene-2-sulfonate, and oxalate.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₃ and R₄are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₅ ishydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₆ and R₇taken together form a double bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂₇ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; and R₂ is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; and R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; and R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), andCH₂O-heterocyclyl; and R₆ and R₇ taken together form a double bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ taken together form a double bond; and R₂₇is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ taken together form a double bond; R₂₇ ishydrogen; and X⁻ is selected from the group consisting of chloride,bromide, iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ taken together form a double bond; R₂₇ ishydrogen; and X⁻ is selected from the group consisting of chloride andbromide.

In one embodiment the present invention provides a pure and isolatedcompound with absolute sterochemistry as shown in formula 2:

or the free base thereof;

wherein independently for each occurrence:

X⁻ is selected from the group consisting of chloride, bromide, iodide,H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate, p-toluenesulfonate,trifluoromethylsulfonate, 10-camphorsulfonate, naphthalene-1-sulfonicacid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, cyclamic acid salt,thiocyanic acid salt, naphthalene-2-sulfonate, and oxalate.

R₁ is hydroxyl or —OC(O)R₈;

R₃ and R₄ are hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl,heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; or R₃ taken together with R₄represent a 4-8 membered optionally substituted heterocyclic ring;

R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₇ is hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, or heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₇ is hydrogen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the stereochemistry of a double bond may be E or Z or a mixture thereof.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, provided thatwhen R₁ is hydroxyl, R₅ is hydrogen, R₆ and R₇ taken together form adouble bond, R₂₇ is hydrogen; R₃ and R₄ are not both hydrogen nor whentaken together represent an unsubstituted azetidine.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₃ isallyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₃ hasformula 9

or the free base thereof;

wherein X₁ ⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₄ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₅ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₆ and R₇taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂₇ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; and R₄ is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ has formula 9

or the free base thereof;

wherein X₁ ⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate; and R₄ is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; R₄ is hydrogen; and R₅ is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ has formula 9

or the free base thereof;

wherein X₁ ⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate; R₄ is hydrogen; and R₅ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; R₄ is hydrogen; R₅ is hydrogen; and R₆ and R₇taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ has formula 9

or the free base thereof;

wherein X₁ ⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate; R₄ is hydrogen; R₅ is hydrogen;and R₆ and R₇ taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; R₄ is hydrogen; R₅ is hydrogen; R₆ and R₇ takentogether form a bond; and R₂₇ is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ has formula 9

or the free base thereof;

wherein X₁ ⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate; R₄ is hydrogen; R₅ is hydrogen; R₆and R₇ taken together form a bond; and R₂₇ is hydrogen.

In one embodiment the present invention provides a pure and isolatedcompound with absolute sterochemistry as shown in formula 3:

wherein X⁻ is selected from the group consisting of chloride, bromide,iodide, H₂PO₄ ⁻, HSO₄ ⁻, methylsulfonate, benzenesulfonate,p-toluenesulfonate, trifluoromethylsulfonate, and 10-camphorsulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, cyclamic acid salt, thiocyanic acid salt,naphthalene-2-sulfonate, and oxalate.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein X⁻ ischloride.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein X⁻ isbromide.

In one embodiment, the present invention relates to a compositioncomprising a compound of any one of the aforementioned compounds and anamino acid.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein theamino acid is selected from the group consisting of:

In one embodiment the present invention provides a compound of formula4:

or a pharmaceutically acceptable salt thereof;

wherein, independently for each occurrence,

W is oxygen or sulfur;

Z is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

n is equal to 0, 1, or 2;

m is equal to 0, 1, or 2;

X and Y are independently C(R₃₀)₂; wherein R₃₀ for each occurrence isindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl; or—[(CR₂)_(p)]—R₁₆;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆;

R₄ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the Formula 4a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉;—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₅ and R₆ are both hydrogen; or R₅ and R₆ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; and

the absolute stereochemistry at a stereogenic center of formula 4 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂₀, R₂₁,R₂₂, R₂₃, R₂₄, R₂₅ are methyl; R₂₆ is hydrogen; Q is a bond; and Z and Ware oxygen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₃ ishydrogen, alkyl, alkenyl, cycloalkyl, aralkyl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₄ ishydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₅ and R₆taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein X and Yare —CH₂—.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein n isequal to 0; and m is equal to 0 or 1.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; and R₂ is hydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; and R₃ is hydrogen, alkyl,alkenyl, cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ is hydrogen, alkyl, alkenyl,cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; and R₄ ishydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrite, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ is hydrogen, alkyl, alkenyl,cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; R₄ is hydrogenor has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; and R₅ and R₆ taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ is hydrogen, alkyl, alkenyl,cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; R₄ is hydrogenor has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₅ and R₆ taken together form a bond; and X and Yare —CH₂—.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ is hydrogen, alkyl, alkenyl,cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; R₄ is hydrogenor has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₅ and R₆ taken together form a bond; X and Y are—CH₂—; n is equal to 0; and m is equal to 0 or 1.

In one embodiment the present invention provides a compound withabsolute sterochemistry as shown in formula 5:

wherein independently for each occurrence:

n is equal to 0, 1, or 2;

m is equal to 0, 1, or 2;

X and Y are independently C(R₃₀)₂; wherein R₃₀ for each occurrence isindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl; or—[(CR₂)_(p)]—R₁₆;

R₁ is hydroxyl or —OC(O)R₈;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl,heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₅ and R₆ are both hydrogen; or R₅ and R₆ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₇ is hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, or heteroaralkyl; and

the stereochemistry of a double bond may be E or Z or a mixture thereof.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₃ isallyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₅ and R₆taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₂₇ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein X and Yare —CH₂—.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein n isequal to 0; and m is equal to 0 or 1.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; and R₃ is allyl.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; and R₅ and R₆ taken together form a bond.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; R₅ and R₆ taken together form a bond; and R₂₇ ishydrogen.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; R₅ and R₆ taken together form a bond; R₂₇ ishydrogen; and X and Y are —CH₂—.

In certain embodiments, the present invention relates to theaforementioned compound and the attendant definitions, wherein R₁ ishydroxyl; R₃ is allyl; R₅ and R₆ taken together form a bond; R₂₇ ishydrogen; X and Y are —CH₂—; n is equal to 0; and m is equal to 0 or 1.

In one embodiment the present invention provides a compound selectedfrom the group consisting of:

The embodiments described above and in the following sections encompasshydroquinone analogs of the geldanamycin family of molecules. Inaddition to reduced forms of 17-AAG(17-allylamino-18,21-dihydro-17-demethoxygeldanamycin), other preferredcompounds of the present invention relates to 18,21-dihydro-geldanamycinfamily including, but not limited to, 18,21-dihydro analogs of17-Amino-4,5-dihydro-17-demethoxy-geldanamycin;17-Methylamino-4,5-dihydro-17-demethoxygeldanamycin;17-Cyclopropylamino-4,5-dihydro-17-demethoxygeldanamycin;17-(2′-Hydroxyethylamino)-4,5-dihydro-17-demethoxygelclanamycin;17-(2-Methoxyethylamino)-4,5-dihydro-17-demethoxygeldanamycin;17-(2′-Fluoroethylamino)-4,5-dihydro-17-demethoxygeldanamycin;17-(S)-(+)-2-Hydroxypropylamino-4,5-dihydro-17-demethoxygeldanamycin;17-Azetidin-1-yl-4,5-dihydro-17-demethoxygeldanamycin;17-(3-Hydroxyazetidin-1-yl)-4,5-dihydro-17-demethoxygeldanamycin;17-Azetidin-1-yl-4,5-dihydro-11-alpha-fluoro-17-demethoxygeldanamycin;17-(2′-Cyanoethylamino)-17-demethoxygeldanamycin;17-(2′-Fluoroethylamino)-17-demethoxygeldanamycin;17-Amino-22-(2′-methoxyphenacyl)-17-demethoxygeldanamycin;17-Amino-22-(3′-methoxyphenacyl)-17-demethoxygeldanetmycin;17-Amino-22-(4′-chlorophenacyl)-17-demethoxygeldanamycin;17-Amino-22-(3′,4′-dichlorophenacyl)-17-demethoxygeldanamycin;17-Amino-22-(4′-amino-3′-iodophenacyl)-17-demethoxygeldanamycin;17-Amino-22-(4′-azido-3′-iodophenacyl)-17-demethoxygeldanamycin;17-Amino-11-alpha-fluoro-17-demethoxygeldanamycin;17-Allylamino-11-alpha-fluoro-17-demethoxygeldanamycin;17-Propargylamino-11-alpha-fluoro-17-demethoxygeldanamycin;17-(2′-Fluoro ethylamino)-11-alpha-fluoro-17-demethoxygeldanamycin;17-Azetidin-1-yl-11-(4′-azidophenyl)sulfamylcarbonyl-17-demethoxygeldanamycin;17-(2′-Fluoroethylamino)-11-keto-17-demethoxygeldanamycin;17-Azetidin-1-yl-11-keto-17-demethoxygeldanamycin; and17-(3′-Hydroxyazetidin-1-yl)-11-keto-17-demethoxygeldanamycin.

It will be understood by one skilled in the art that the methodologyoutlined herein can be used with any amino substituted benzoquinoneansamycin.

The compositions of the present invention exists as salts of the reducedansamycin, e.g., HCl or H₂SO₄ salts. In another embodiment the compoundsare co-crystallized with another salt, such as an amino acid, e.g.,glycine. In general, in these embodiments, the ratio of amino acid toansamycin can vary, but is preferably from 2:1 to 1:2 aminoacid:ansamycin.

Compositions & Formulations

The present invention also provides a pharmaceutical compositioncomprising any one of the aforementioned compounds and at least onepharmaceutically acceptable excipient.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising: at least one pharmaceutically acceptableexcipient; and a compound of formula 6:

or the free base thereof;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 6a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(R₁₉), and—P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 6 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, provided thatwhen R₁ is hydroxyl, R₂ is hydrogen, R₅ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a buffering agent.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant; and a buffering agent.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant; and a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a buffering agent; and a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant; a buffering agent; and a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidantioxidant is ascorbate, cysteine hydrochloride, sodium bisulfate,sodium metabisulfite, sodium sulfite, thioglycerol, sodiummercaptoacetate, sodium formaldehyde sulfoxylate, ascorbyl palmitate,butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propylgallate, or alpha-tocopherol.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidantioxidant is ascorbate.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidbuffering agent is citrate, ascorbate, phosphate, bicarbonate,carbonate, fumarate, acetate, tartarate, malate, succinate, lactate,maleate, glycine, or other naturally-occurring α- or, β-amino acids.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidbuffering agent is citrate.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidmetal chelator is citric acid, ethylenediamine tetraacetic acid (EDTA)and its salt, DTPA (diethylene-triamine-penta-acetic acid) and its salt,EGTA and its salt, NTA (nitriloacetic acid) and its salt, sorbitol andits salt, tartaric acid and its salt, N-hydroxy iminodiacetate and itssalt, hydroxyethyl-ethylene diamine-tetraacetic acid and its salt, 1-and 3-propanediamine tetra acetic acid and their salts, 1- and3-diamino-2-hydroxy propane tetra-acetic acid and their salts, sodiumgluconate, hydroxy ethane diphosphonic acid and its salt, or phosphoricacid and its salt.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidmetal chelator is EDTA.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidbuffering agent is citrate, said antioxidant is ascorbate, and saidmetal chelator is EDTA.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.001 to about 0.1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.01 to about 0.05.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said ascorbic acid to said compound of formula 6 is inthe range from about 0.001 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said ascorbic acid to said compound of formula 6 is inthe range from about present 0.01 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said citrate to said compound of formula 6 is in therange of about 0.05 to about 2.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said citrate to said compound of formula 6 is in therange of about 0.2 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.001 to about 0.1; and the molar ratio of said ascorbic acidto said compound of formula 6 is in the range from about 0.001 to about1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.01 to about 0.05; and the molar ratio of said ascorbic acidto said compound of formula 6 is in the range from about present 0.01 toabout 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.001 to about 0.1; the molar ratio of said ascorbic acid tosaid compound of formula 6 is in the range from about 0.001 to about 1;the molar ratio of said citrate to said compound of formula 6 is in therange of about 0.05 to about 2.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.01 to about 0.05; the molar ratio of said ascorbic acid tosaid compound of formula 6 is in the range from about present 0.01 toabout 1; the molar ratio of said citrate to said compound of formula 6is in the range of about 0.2 to about 1.

In certain embodiments, the present invention relates to apharmaceutical composition of any one of the aforementionedcompositions, further comprising a solubilizing agent.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidsolubilizing agent is polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, benzyl alcohol, ethyl alcohol, polyethyleneglycols, propylene glycol, glycerin, cyclodextrin, or poloxamers.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising: at least one pharmaceutically acceptableexcipient; a compound of formula 6:

or the free base thereof; and a compound of formula 10, wherein saidcompound of formula 10 is present in the range of about 0.00001% toabout 5% (m/v):

or pharmaceutically acceptable salt thereof;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 6a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₈), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(OR₁₉),and —P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 6 or 10may be R or S or a mixture thereof and the stereochemistry of a doublebond may be E or Z or a mixture thereof.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, provided thatwhen R₁ is hydroxyl, R₂ is hydrogen, R₅ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a buffering agent.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant; and a buffering agent.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant; and a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a buffering agent; and a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising an antioxidant; a buffering agent; and a metal chelator.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidantioxidant is ascorbate, cysteine hydrochloride, sodium bisulfate,sodium metabisulfite, sodium sulfite, thioglycerol, sodiummercaptoacetate, sodium formaldehyde sulfoxylate, ascorbyl palmitate,butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propylgallate, or alpha-tocopherol.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidantioxidant is ascorbate.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidbuffering agent is citrate, ascorbate, phosphate, bicarbonate,carbonate, fumarate, acetate, tartarate, malate, succinate, lactate,maleate, glycine, or other naturally-occurring α- or β-amino acids.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidbuffering agent is citrate.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidmetal chelator is citric acid, ethylenediamine tetraacetic acid (EDTA)and its salt, DTPA (diethylene-triamine-penta-acetic acid) and its salt,EGTA and its salt, NTA (nitriloacetic acid) and its salt, sorbitol andits salt, tartaric acid and its salt, N-hydroxy iminodiacetate and itssalt, hydroxyethyl-ethylene diamine-tetraacetic acid and its salt, 1-and 3-propanediamine tetra acetic acid and their salts, 1- and3-diamino-2-hydroxy propane tetra-acetic acid and their salts, sodiumgluconate, hydroxy ethane diphosphonic acid and its salt, or phosphoricacid and its salt.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidmetal chelator is EDTA.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidbuffering agent is citrate, said antioxidant is ascorbate, and saidmetal chelator is EDTA.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.001 to about 0.1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.01 to about 0.05.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said ascorbic acid to said compound of formula 6 is inthe range from about 0.001 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said ascorbic acid to said compound of formula 6 is inthe range from about present 0.01 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said citrate to said compound of formula 6 is in therange of about 0.05 to about 2.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said citrate to said compound of formula 6 is in therange of about 0.2 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.001 to about 0.1; and the molar ratio of said ascorbic acidto said compound of formula 6 is in the range from about 0.001 to about1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.01 to about 0.05; and the molar ratio of said ascorbic acidto said compound of formula 6 is in the range from about present 0.01 toabout 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.001 to about 0.1; the molar ratio of said ascorbic acid tosaid compound of formula 6 is in the range from about 0.001 to about 1;the molar ratio of said citrate to said compound of formula 6 is in therange of about 0.05 to about 2.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein themolar ratio of said EDTA to said compound of formula 6 is in the rangefrom about 0.01 to about 0.05; the molar ratio of said ascorbic acid tosaid compound of formula 6 is in the range from about present 0.01 toabout 1; the molar ratio of said citrate to said compound of formula 6is in the range of about 0.2 to about 1.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, furthercomprising a solubilizing agent.

In certain embodiments, the present invention relates to theaforementioned composition and the attendant definitions, wherein saidsolubilizing agent is polyoxyethylene sorbitan fatty acid esters,polyoxyethylene stearates, benzyl alcohol, ethyl alcohol, polyethyleneglycols, propylene glycol, glycerin, cyclodextrin, or poloxamers.

In certain embodiments, the present invention relates to apharmaceutical composition of any one of the aforementionedcompositions, wherein said compound of formula 6 is present at aconcentration of about 0.00016 M to about 0.160 M.

In certain embodiments, the present invention relates to apharmaceutical composition of any one of the aforementionedcompositions, wherein said compound of formula 6 is present at aconcentration of about 0.00032 M to about 0.080 M.

Methods of Making

A variety of methodologies can be adapted for generating the compoundsof the present invention. In general, the steps involve (1) convertingthe ansamycin to a 17-demethoxy-17-amino analog (e.g., 17-AAG), (2)reducing the benzoquinone in the ansamycin to give a hydroquinone, and(3) treating said hydroquinone with a Bronsted acid, thereby providing acompound of the present invention.

A benzoquinone-containing macrocyclic molecule, can be obtained viafermentation of a strain producing the compound (for example, see WO03/072794 and U.S. Pat. No. 3,595,955). Alternatively, synthetic orsemi-synthetic methodology can be used to produce the ansamycin (seeU.S. Pat. No. 5,387,584 and WO 00/03737). Further, there are commercialsuppliers of isolated fermentation materials, such as geldanamycin;therefore, such materials are readily available.

In preferred embodiments, synthetic methodology is used to createanalogs of a natural product isolated from an organism using knownmethods. For example, geldanamycin is isolated from a fermentationculture of an appropriate micro-organism and may be derivatized using avariety of functionalization reactions known in the art. Representativeexamples include metal-catalyzed coupling reactions, oxidations,reductions, reactions with nucleophiles, reactions with electrophiles,pericyclic reactions, installation of protecting groups, removal ofprotecting groups, and the like. Many methods are known in the art forgenerating analogs of the various benzoquinone ansamycins (for examples,see U.S. Pat. Nos. 4,261,989; 5,387,584; and 5,932,566 and J. Med. Chem.1995, 38, 3806-3812, herein incorporated by reference). These analogsare readily reduced, using methods outlined below, to yield the18,21-dihydro derivatives of the present invention.

Once the starting material is obtained, the benzoquinone is reduced toform a hydroquinone and then reacted with an acid, for instance HCl, togenerate a C-17 ammonium hydroquinone ansamycin in an air-stable saltform. In an alternate embodiment the hydroquinone free base is reactedwith an acid halide of an amino acid in place of a Bronsted acid togenerate air-stable C-17 ammonium hydroquinone ansamycin co-saltderivatives. This method is exemplified in Example 3.

A variety of methods and reaction conditions can be used to reduce thebenzoquinone portion of the ansamycin. Sodium hydrosulfite may be usedas the reducing agent. Other reducing agents that can be used include,but are not limited to, zinc dust with acetic anhydride or acetic acid,ascorbic acid and electrochemical reductions.

Reduction of the benzoquinone moiety of the ansamycin derivative may beaccomplished using sodium hydrosulfite in a biphasic reaction mixture.Typically, the geldanamycin analog is dissolved in an organic solvent,such as EtOAc. Other solvents that can be used include, but are notlimited to, dichloromethane, chloroform, dichloroethane, chlorobenzene,THF, MeTHF, diethyl ether, diglyme, 1,2-dimethoxyethane, MTBE, THP,dioxane, 2-ethoxybutane, methyl butyl ether, methyl acetate, 2-butanone,water and mixtures thereof. Two or more equivalents of sodiumhydrosulfite are then added as a solution in water (5-30% (m/v),preferably 10% (m/v)), to the reaction vessel at room temperature.Aqueous solutions of sodium hydrosulfite are unstable and therefore needto be freshly prepared just prior to use. Vigorous mixing of thebiphasic mixture ensures reasonable reaction rates.

The reaction can readily be followed at this step by visual inspectionsince the starting material 17-AAG has a purple color which willdisappear as the reaction proceeds to the product dihydro-17AAG, whichis yellow. However, HPLC/UV or other analytical methods can be used tomonitor the reaction.

Upon completion of the reduction, the crude reaction mixture product maybe used in the next step without purification to minimize oxidation ofthe hydroquinone. However, purification, preferably byrecrystallization, can be performed if the conditions are monitored tomaintain the reduced form of the benzoquinone.

The hydroquinone-containing ansamyacin is unstable and, in the presenceof small amounts of oxygen or other oxidants, the hydroquinone moietymay be rapidly oxidized to the quinone species. Remarkably, thehydroquinone can be converted into an air-stable species by reactionwith an acid, or by reaction with an acid halide of an amino acid. Inthe examples, the C-17 allyl amino group is protonated to generate avariety of air-stable C-17 ammonium salt hydroquinone geldanamycinanalogs. In addition, the C-17 ammonium salt hydroquinones formed havethe added benefit of being highly soluble in aqueous solutions (>200mg/mL), unlike 17-AAG (<100 μg/mL).

The ammonium salt hydroquinone is formed by the addition of a solutionof an acid, such as HCl, in an organic solvent, such as EtOAc, DCM, IPAor dioxane, to the hydroquinone containing ansamycin in an organicsolution; the organic solvents may be independently acetone,dichloromethane, chloroform, dichloroethane, chlorobenzene, THF, MeTHF,diethyl ether, diglyme, 1,2-dimethoxyethane, MTBE, THP, dioxane,2-ethoxybutane, methyl butyl ether, methyl acetate, 2-butanone, undernitrogen.

The ammonium salt of the hydroquinone is collected by filtration incases where the product precipitates from solution. In cases where theammonium salt hydroquinone does not precipitate, the reaction solutionis concentrated under reduced pressure to yield the product.

A variety of air-stable ammonium salt hydroquinone ansamycins can besynthesized by using organic or inorganic acids. Some acids that can beused include, but are not limited to HCl, HBr, H₂SO₄, methansulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid, triflic acid,camphorsulfonic acid, naphthalene-1,5-disulfonic acid,ethan-1,2-disulfonic acid, cyclamic acid, thiocyanic acid,naphthalene-2-sulfonic acid, oxalic acid, and the like. See, forexample, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19. The acid used preferably should have a pKa sufficient toprotonate the aniline nitrogen. Thus, any acid with a pKa between about−10 and about 7, preferably about −10 and about 4, more preferablybetween about −10 and about 1, and even more preferably between about−10 and about −3 may be used to generate the ammonium salt hydroquinone.

The present invention further provides methods for recrystallizing thecompounds of the present invention. In such methods, recrystallizationis accomplished by dissolving the compound in the minimal amount of aninert polar organic solvent, such as MeOH, EtOH, or IPA, and slowlyadding a miscible organic solvent, such as an aliphatic ether, ethylacetate, methyl acetate, chloroform or DCM, causing the solution tobecome turbid. The mixture is then allowed to sit for a suitable periodof time, and optionally cooled, and the resulting solid is collected byfiltration, washed and dried under reduced pressure.

One aspect of the invention relates to a method of preparing a compound,comprising: combining a compound of formula 7 with a reducing agent in areaction solvent to give a compound of formula 8; and

combining said compound of formula 8 with a pharmaceutically acceptableacid to give said compound of formula 1;

wherein independently for each occurrence:

W is oxygen or sulfur;

Q is oxygen, NR, N(acyl) or a bond;

X⁻ is a conjugate base of a pharmaceutically acceptable acid;

R for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl;

R₁ is hydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—;

R₃ and R₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₃ takentogether with R₄ represent a 4-8 membered optionally substitutedheterocyclic ring;

R₅ is selected from the group consisting of H, alkyl, aralkyl, and agroup having the formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl;

R₆ and R₇ are both hydrogen; or R₆ and R₇ taken together form a bond;

R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆;

R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₀ and R₁₁ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆;

R₁₃ and R₁₄ are each independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, and —[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄taken together with the nitrogen to which they are bonded represent a4-8 membered optionally substituted heterocyclic ring;

R₁₆ for each occurrence is independently selected from the groupconsisting of hydrogen, hydroxyl, acylamino, —N(R₁₈)COR₁₉,—N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉), —OC(O)N(R₁₈)(R₁₉),—SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈, —C(O)N(OH)(R₁₈),—OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉), —N(R₁₈)P(O)(OR₁₈)(OR₁₉),and —P(O)(OR₁₈)(OR₁₉);

p is 1, 2, 3, 4, 5, or 6;

R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

R₁₉ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; or R₁₈ taken together with R₁₉represent a 4-8 membered optionally substituted ring;

R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for each occurrence are independentlyalkyl;

R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈, or —CH(OR₁₈)₂;

R₂₆ and R₂₇ for each occurrence are independently selected from thegroup consisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl;

provided that when R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ takentogether form a double bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ ismethyl, R₂₃ is methyl, R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen,R₂₇ is hydrogen, Q is a bond, and W is oxygen; R₃ and R₄ are not bothhydrogen nor when taken together represent an unsubstituted azetidine;and

the absolute stereochemistry at a stereogenic center of formula 1 may beR or S or a mixture thereof and the stereochemistry of a double bond maybe E or Z or a mixture thereof.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said reducing agent is sodium hydrosulfite, zinc,ascorbic acid, or an electrochemical reduction.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said reducing agent is sodium hydrosulfite.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said reaction solvent is dichloromethane, chloroform,dichloroethane, chlorobenzene, THF, 2-MeTHF, diethyl ether, diglyme,1,2-dimethoxyethane, MTBE, THP, dioxane, 2-ethoxybutane, methyl butylether, ethyl acetate, methyl acetate, 2-butanone, water or mixturesthereof.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said reaction solvent is a mixture of ethyl acetate andwater.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid has a pKa between about −10 and about 7 inwater.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid has a pKa between about −10 and about 4 inwater.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid has a pKa between about −10 and about 1 inwater.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid has a pKa between about −10 and about −3 inwater.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid is HCl, HBr, H₂SO₄, methansulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, triflic acid,camphorsulfonic acid, naphthalene-1,5-disulfonic acid,ethan-1,2-disulfonic acid, cyclamic acid, thiocyanic acid,naphthalene-2-sulfonic acid, or oxalic acid.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid is HCl.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid is HBr.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid is added as a gas.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said acid is dissolved in an organic solvent.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said organic solvent is EtOAc, DCM, IPA or dioxane, tothe hydroquinone containing ansamycin in an organic solution, such asacetone, dichloromethane, chloroform, dichloroethane, chlorobenzene,THF, 2-MeTHF, diethyl ether, diglyme, 1,2-dimethoxyethane, MTBE, THP,dioxane, 2-ethoxybutane, methyl butyl ether, methyl acetate, or2-butanone.

In one embodiment, the present invention relates to the aforementionedmethod, wherein R₁ is hydroxyl; R₂ is hydrogen; R₃ is allyl; R₄ ishydrogen; R₅ is H; R₆ and R₇ taken together form a bond; R₂₀ is methyl;R₂₁ is methyl; R₂₂ is methyl; R₂₃ is methyl; R₂₄ is methyl; R₂₅ ismethyl; R₂₆ is hydrogen; R₂₇ is hydrogen; W is oxygen; and Q is a bond.

Pharmaceutical Compositions

When the compounds of the Formula 1 and 3 and their pharmaceuticallyacceptable salts are used as antiproliferative agents, such asanticancer agents, they can be administered to a mammalian subjecteither alone or in combination with pharmaceutically acceptable carriersor diluents in a pharmaceutical composition according to standardpharmaceutical practice. The compounds can be administered orally orparenterally, preferably parenterally. Parenteral administrationincludes intravenous, intramuscular, intraperitoneal, subcutaneous andtopical, the preferred method being intravenous administration.

Accordingly, the present invention provides pharmaceutically acceptablecompositions which comprise a therapeutically-effective amount of one ormore of the compounds described above (Formula 1 and 3), formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. The pharmaceutical compositions of thepresent invention may be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; and (2) oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue. The preferred method of administration ofcompounds of the present invention is parental administration(intravenous).

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

The pharmaceutically acceptable salts of the compounds of the presentinvention include the conventional nontoxic salts or quaternary ammoniumsalts of the compounds, e.g., from non-toxic organic or inorganic acids.For example, such conventional nontoxic salts include those derived frominorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives, solubilizing agents, buffers and antioxidants can also bepresent in the compositions.

Examples of pharmaceutically-acceptable antioxidants include, but arenot limited to: (1) water soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite, thioglycerol, sodium mercaptoacetate, and sodium formaldehydesulfoxylate; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol.

Examples of pharmaceutically-acceptable buffering agents include, butare not limited to citrate, ascorbate, phosphate, bicarbonate,carbonate, fumarate, acetate, tartarate and malate.

Examples of pharmaceutically-acceptable solubilizing agents include, butare not limited to polyoxyethylene sorbitan fatty acid esters (includingpolysorbate 80), polyoxyethylene stearates, benzyl alcohol, ethylalcohol, polyethylene glycols, propylene glycol, glycerin, cyclodextrin,and poloxamers.

Examples of pharmaceutically-acceptable complexing agents include, butare not limited to, cyclodextrins (alpha, beta, gamma), especiallysubstituted beta cyclodextrins such as 2-hydroxypropyl-beta, dimethylbeta, 2-hydroxyethyl beta, 3-hydroxypropyl beta, trimethyl beta.

Examples of pharmaceutically-acceptable metal chelating agents include,but are not limited to, citric acid, ethylenediamine tetraacetic acid(EDTA) and its salt, DTPA (diethylene-triamine-penta-acetic acid) andits salt, EGTA and its salt, NTA (nitriloacetic acid) and its salt,sorbitol and its salt, tartaric acid and its salt, N-hydroxyiminodiacetate and its salt, hydroxyethyl-ethylene diamine-tetraaceticacid and its salt, 1- and 3-propanediamine tetra acetic acid and theirsalts, 1- and 3-diamino-2-hydroxy propane tetra-acetic acid and theirsalts, sodium gluconate, hydroxy ethane diphosphonic acid and its salt,and phosphoric acid and its salt.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers (liquid formulation), liquid carriers followed bylyophylization (powder formulation for reconstitution with sterile wateror the like), or finely divided solid carriers, or both, and then, ifnecessary, shaping or packaging the product.

Pharmaceutical compositions of the present invention suitable forparenteral administration comprise one or more compounds of theinvention in combination with one or more pharmaceutically-acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain sugars, alcohols, antioxidants, buffers,bacteriostats, chelating agents, solutes which render the formulationisotonic with the blood of the intended recipient or suspending orthickening agents. In the examples, the active ingredients are broughttogether with the pharmaceutically acceptable carriers in solution andthen lyophilized to yield a dry powder. The dry powder is packaged inunit dosage form and then reconstituted for parental administration byadding a sterile solution, such as water or normal saline, to thepowder.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the compounds of the present inventionmay be ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

One preferred formulation for the compounds of the present invention isan aqueous buffer containing citric acid (from about 5 mM to about 250mM, preferably from about 25 mM to about 150 mM), ascorbic acid (fromabout 0.1 mM to about 250 mM, preferably from about 0.1 mM to about 50mM), and edetate (calcium-disodium ethylenediamine tetraacetic acid,EDTA, from about 0.2 mM to about 20 mM, preferably from about 1 mM toabout 3 mM) with the pH being adjusted to about 3.1 with sodiumhydroxide. The components of the formulation act as buffering agent,anti-oxidant and metal chelator, respectively.

It is important for formulations of the compounds of the presentinvention to provide solubility and redox stability to this hydroquinonesalt. Compounds of the present invention are significantly solubilizedat lower pH when the amine is protonated. The species distribution isimportant since the ionized form is more soluble while the free base(un-ionized form) is less soluble. Therefore a formulation will optimizethe solubility by controlling the pH of the solution. A buffering agentsuch as citrate which has a high buffering capacity at a preferred pHrange is one such preferred formulation component. Preferably bufferingagents will buffer the formulation between a pH of about 1.5 to about5.0, more preferably between a pH of about 1.8 to about 3.5, and evenmore preferably between a pH of about 3 to about 3.3.

The hydoquinone analogs of the present invention may oxidize onprolonged standing in solution. Heavy metals, such as iron and copper,are capable of catalyzing oxidation reactions and can be found in tracequantities in typical reagents and labware. Protection from theoxidizing nature of heavy metals can be afforded by metal chelators suchas EDTA (ethylene diamine tetraacetic acid). Other known chelators are,for example, citric acid, DTPA (diethylene-triamine-penta-acetic acid)and its salt, EGTA and its salt, NTA (nitriloacetic acid) and its salt,sorbitol and its salt, tartaric acid and its salt, N-hydroxyiminodiacetate and its salt, hydroxyethyl-ethylene diamine-tetraaceticacid and its salt, 1- and 3-propanediamine tetra acetic acid and theirsalts, 1- and 3-diamino-2-hydroxy propane tetra-acetic acid and theirsalts, sodium gluconate, hydroxy ethane diphosphonic acid and its salt,and phosphoric acid and its salt.

Another important method of preventing oxidation is to add ananti-oxidant. One preferred anti-oxidant is ascorbic acid (ascorbate).This reagent protects compounds from the oxidizing effect of molecularoxygen dissolved in aqueous media. In certain embodiments, ascorbate isused as a component in formulations of the hydroquinone analogs of thepresent invention.

The formulations of the present invention include formulations that arecapable of shelf storage as well as formulations used for directadministrations to a patient. Specifically, the pharmaceuticalcompositions/formulations of the present invention are provide in a formmore concentrated than that suitable for direct administration to apatient. Such a composition is typically diluted into and IV bag foradministration to a patient.

It is important in such a use that the formulation contained in the IVbag be stable for from about 5 minutes to about 2 hours, more preferablystable for about 1 hour to about 2 hours, most preferably stable forabout 2 hours. Stability needs to be maintained throughout the period inwhich the drug is administered.

Further, it is important that the buffering capacity of the diluted IVbag formulation be sufficient to achieve this stability while not beingtoo high of a concentration to cause an adverse reaction in the patient.Too much buffer being present may result in a number of undesirableeffects on the patient.

Methods of Therapy and Treatment

The present invention provides water soluble hydroquinone containingcompounds that rapidly oxidize to 17-amino substituted benzoquinonegeldanamycin analogs (e.g. 17-AAG) in vitro and in vivo at physiologicalpH. As such, the hydroquinone analogs of the present invention exhibitsimilar biological activities and therapeutic profiles as do 17-aminosubstituted geldanamycin analogs and may be used for all knowntherapeutic indications that 17-amino substituted geldanamycin analogsare useful in treating. 17-amino substituted geldanamycin analogs, andin particular 17-AAG, are highly potent and selective inhibitors ofHSP90.

The present invention further provides methods for treating,ameliorating one or more of the symptoms of, and reducing the severityof hyperpoliferative disorders, ie cancer, as well as other HSP90mediated disorders or conditions. Since the compositions of the presentinvention are more soluble than the oxidized benzoquinone forms, thecompositions are more easily administered resulting in better clinicaloutcomes for any of the known uses of the parent molecules.

The methods of treatment of the present invention involve administeringa therapeutically effective amount of a compound of the presentinvention to a subject suffering from an HSP90 mediated disorder orcondition, such as cancer. Descriptions of the compositions,formulations, dosing, modes of administration and treatment aredescribed herein.

Certain 17-amino substituted analogs of geldanamycin have beensynthesized, and their use as antitumor agents is described in U.S. Pat.Nos. 4,261,989 and 5,387,584, 5,932,566 and published PCT applicationsWO 00/03737 and WO 03/072794 (incorporated herein by reference).Structure activity relationships of 17-amino substituted geldanamycinanalogs have shed more light on the chemical features required forinhibition of HSP90 (See, e.g., J. Med. Chem. (1995) 38:3806-3812, J.Med. Chem. (1995) 38:3813-3820, and Clin. Cancer Res. (1999) 5:3781).

Among the more successful 17-amino substituted geldamaycin analogs is17-AAG, which has shown broad antitumor activity in vitro and in vivoand is currently in multiple phase I/II clinical trials. 17-AAG exhibitsdifferential cytoxicity against a broad range of tumor types in the NCI60 tumor cell line panel. The mean IC₅₀ over all cell lines in the panelis 120 nM (Developmental Therapeutics Program Website:http://dtp.nci.nih.gov/, mean graph for compound S330507).

In addition, 17-AAG has been shown to have activity against a number ofcell lines, including, but not limited to, melanoma (Anti-Cancer Drugs(2004) 15: 377-388), prostate cancer (Clin. Cancer Res. (2002) 8:986-993), breast cancer (Cancer. Res. (2001) 61: 2945-2952), non-smallcell lung cancer (Ann. Thorac. Surg. (2000) 70: 1853-1860), leukemias(Cancer Res. (2001) 61: 1799-1804), and colon cancer (J. Natl. CancerInst. (2003) 95: 1624-1633).

In one embodiment, the present invention provides a method of treatingcancer, comprising administering to a mammal in need thereof atherapeutically effective amount of anyone of the aforementionedcompounds; or a therapeutically effective amount of anyone of theaforementioned pharmaceutical compositions.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said cancer is a cancer of the hematopoietic system,immune system, endocrine system, pulmonary system, gastrointestinalsystem, musculoskeletal system, reproductive system, central nervoussystem, or urologic system.

In one embodiment, the present invention relates to the aforementionedmethod, wherein the cancer is located in the mammal's myeloid tissues,lymphoid tissues, pancreatic tissues, thyroid tissues, lungs, colontissues, rectal tissues, anal tissues, liver tissues, skin, bone,ovarian tissues, uterine tissues, cervical tissues, breast, prostate,testicular tissues, brain, brainstem, meningeal tissues, kidney, orbladder.

In one embodiment, the present invention relates to the aforementionedmethod, wherein the cancer is located in the mammal's myeloid tissues,lymphoid tissues, breast, lung, ovary, or prostate.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said cancer is breast cancer, multiple myeloma, prostatecancer, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronicmyeloid leukemia, renal cell carcinoma, malignant melanoma, pancreaticcancer, lung cancer, colorectal carcinoma, colon cancer, brain cancer,renal cancer, head and neck cancer, bladder cancer, thyroid cancer,prostate cancer, ovarian cancer, cervical cancer, or myelodysplasticsyndrome.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said mammal's cancer is breast cancer, acute myeloidleukemia, chronic myeloid leukemia, melanoma, multiple myeloma, lungcancer, ovarian cancer, or prostate cancer.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said mammal is a primate, equine, canine, feline, orbovine.

In one embodiment, the present invention relates to the aforementionedmethod, wherein said mammal is a human.

In one embodiment, the present invention relates to the aforementionedmethod, wherein the mode of administration of said compound isinhalation, oral, intravenous, sublingual, ocular, transdermal, rectal,vaginal, topical, intramuscular, intra-arterial, intrathecal,subcutaneous, buccal, or nasal.

In one embodiment, the present invention relates to the aforementionedmethod, wherein the mode of administration is intravenous.

Combination Therapy

In another embodiment, the present invention provides methods oftreatment wherein the compounds and compositions of the invention areused at sub-cytotoxic levels in combination with at least one otheragent in order to achieve selective activity in the treatment of cancer.In certain embodiments, the compounds of the present invention are usedto reduce the cellular levels of properly folded HSP90 client proteins,which are then effectively inhibited by the second agent or whosedegradation in the proteasome is inhibited using a proteasome inhibitor,e.g., Velcade™. Binding of the client proteins to HSP90 stabilizes theclient proteins and maintains them in a soluble, inactive form ready torespond to activating stimuli. Binding of a benzoquinone ansamycinanalog of the present invention to HSP90 results in targeting of theclient protein to the proteasome, and subsequent degradation. Using anagent that targets and inhibits the proteasome blocks proteasomedegradation leading to increased in cellular apoptosis and cell death.

Some examples of antineoplastic agents which can be used in combinationwith the methods of the present invention include, in general,alkylating agents; anti-angiogenic agents; anti-metabolites;epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes;anti-mitotics; biological response modifiers and growth inhibitors;hormonal/anti-hormonal therapeutic agents and haematopoietic growthfactors.

Exemplary classes of antineoplastic agents further include theanthracycline family of drugs, the vinca drugs, the mitomycins, thebleomycins, the cytotoxic nucleosides, the epothilones, discodermolide,the pteridine family of drugs, diynenes and the podophyllotoxins.

Particularly useful members of those classes include, for example,carminomycin, daunorubicin, aminopterin, methotrexate, methopterin,dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin orpodophyllotoxin derivatives such as etoposide, etoposide phosphate orteniposide, melphalan, vinblastine, vincristine, leurosidine, Velcade,doxorubicin, vindesine, leurosine, imatinib mesylate, paclitaxel, taxol,and the like. In a preferred embodiment, the antineoplastic agent isVelcade, doxorubicin, taxotere, docetaxel, paclitaxel, cis-platin,imatinib mesylate, or gemcitebine. In a preferred embodiment, theantineoplastic agent is Velcade or doxorubicin.

Other useful antineoplastic agents include estramustine, carboplatin,cyclophosphamide, bleomycin, gemcitibine, ifosamide, melphalan,hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate,dacarbazine, L-asparaginase, camptothecin, CPT-11, topotecan, ara-C,bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives,interferons and interleukins.

The chemotherapeutic agent and/or radiation therapy can be administeredaccording to therapeutic protocols well known in the art. It will beapparent to those skilled in the art that the administration of thechemotherapeutic agent and/or radiation therapy can be varied dependingon the disease being treated and the known effects of thechemotherapeutic agent and/or radiation therapy on that disease. Also,in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents (i.e., antineoplastic agent or radiation) on thepatient, and in view of the observed responses of the disease to theadministered therapeutic agents.

Also, in general, compounds of the present invention and thechemotherapeutic agent do not have to be administered in the samepharmaceutical composition, and may, because of different physical andchemical characteristics, have to be administered by different routes.For example, compounds of the present invention may be administeredintravenously to generate and maintain good blood levels, while thechemotherapeutic agent may be administered orally. The determination ofthe mode of administration and the advisability of administration, wherepossible, in the same pharmaceutical composition, is well within theknowledge of the skilled clinician. The initial administration can bemade according to established protocols known in the art, and then,based upon the observed effects, the dosage, modes of administration andtimes of administration can be modified by the skilled clinician.

The particular choice of chemotherapeutic agent or radiation will dependupon the diagnosis of the attending physicians and their judgment of thecondition of the patient and the appropriate treatment protocol.

A compound of the present invention, and chemotherapeutic agent and/orradiation may be administered concurrently (e.g., simultaneously,essentially simultaneously or within the same treatment protocol) orsequentially, depending upon the nature of the proliferative disease,the condition of the patient, and the actual choice of chemotherapeuticagent and/or radiation to be administered in conjunction (i.e., within asingle treatment protocol) with a compound of the present invention.

If a compound of the present invention, and the chemotherapeutic agentand/or radiation are not administered simultaneously or essentiallysimultaneously, then the optimum order of administration of the compoundof the present invention, and the chemotherapeutic agent and/orradiation, may be different for different tumors. Thus, in certainsituations the compound of the present invention may be administeredfirst followed by the administration of the chemotherapeutic agentand/or radiation; and in other situations the chemotherapeutic agentand/or radiation may be administered first followed by theadministration of a compound of the present invention. This alternateadministration may be repeated during a single treatment protocol. Thedetermination of the order of administration, and the number ofrepetitions of administration of each therapeutic agent during atreatment protocol, is well within the knowledge of the skilledphysician after evaluation of the disease being treated and thecondition of the patient. For example, the chemotherapeutic agent and/orradiation may be administered first, especially if it is a cytotoxicagent, and then the treatment continued with the administration of acompound of the present invention followed, where determinedadvantageous, by the administration of the chemotherapeutic agent and/orradiation, and so on until the treatment protocol is complete.

Thus, in accordance with experience and knowledge, the practicingphysician can modify each protocol for the administration of a component(therapeuticagent, i.e., compound of the present invention,chemotherapeutic agent or radiation) of the treatment according to theindividual patient's needs, as the treatment proceeds.

Dosage

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or salt thereof, the route of administration, thetime of administration, the rate of excretion or metabolism of theparticular compound being employed, the rate and extent of absorption,the duration of the treatment, other drugs, compounds and/or materialsused in combination with the particular compound employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable dose of a compound of the invention will be thatamount of the compound which is the lowest safe and effective dose toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous doses ofthe compounds of the present invention for a patient will range fromabout 10 mg to about 1000 mg per meter² dosed twice per week, preferablybetween about 75 mg to 750 mg per meter² dosed twice per week, and evenmore preferably 100 mg to 500 mg per meter² dosed twice per week.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

One or more other active compounds may be added to the formulationsdescribed above to provide formulations for combination cancer therapy.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.Further, the amino acid are represented in zwitterionic form and canalso be further protonated and exist as the salt.

Example 1 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1a) (1.0 equiv) is dissolved in dichloromethane(0.02 M) and stirred with a 10% aqueous solution of sodium hydrosulfite(1:1; DCM:aqueous solution). The solution is stirred for 30 minutes. Theorganic layer is then removed via syringe and the aqueous solution isextracted once more with dichloromethane. The combined organic solutionsare washed with brine and then added directly to a solution of an acidchloride (1.0 equiv) in dichloromethane (0.001 M). The reaction mixtureis stirred for 12 h and poured into a solution of dichloromethane. Theorganic layer is then washed with additional water (2.0 mL); thecombined aqueous layers are then lyophilized to yield the product.

Example 2 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1 a) (0.25 mmol, 1.0 equiv) is dissolved indichloromethane (3 mL) and stirred with a 10% aqueous solution of sodiumhydrosulfite (1.5 mL). The solution is stirred for 30 minutes. Theorganic layer is then removed via syringe and the aqueous solution isextracted once more with dichloromethane. The combined organic solutionsare diluted with 3 mL of EtOAc, washed with brine and further dried byazeotropic removal of residual water and EtOAc under reduced pressure (3mL of solvent total removed under reduced pressure). To this solution isadded a solution of an acid in an organic solvent. The resultingsolution is then cooled to −5° C. and an acid (0.25 mmol) in toluene isadded (0.2 mL). A solid slowly crashes out of solution. MTBE (3 mL) isthen added and the resulting mixture is allowed to warm to RT and isstirred at this temperature for 50 minutes. The solid is then collectedby vacuum filtration, is washed with MTBE (2×3 mL), and is dried underreduced pressure to yield the product.

Example 3 Preparation of Dimethylamino Acetate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (9.1 mg, 0.016 mmol, 1.0 equiv) wasdissolved in 1.0 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (1.0 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of dimethylaminoacetyl acid chloridehydrochloride (2.5 mg, 0.016 mmol, 1.0 equiv) in 0.20 mLdichloromethane. The reaction mixture was stirred for 2 h and pouredinto a separatory funnel with 3.0 mL water. The organic layer wasextracted and then washed with additional 2.0 mL water. The combinedaqueous layers were lyophilized to yield 2 as a white fluffy powder (7.1mg, 0.011 mmol, 66% yield). The material was analyzed by ¹H NMR in D₂Oand LC-MS.

Example 4 Preparation of α-Aminoisobutyrate Co-Salt of the Hydroquinoneof 17-AAG

17-Allylaminogeldanamycin (1) (16.7 mg, 0.0285 mmol, 1.0 equiv) wasdissolved in 1.5 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (1.5 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of acid chloride hydrochloride (4.4 mg,0.0314 mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 3 asa white fluffy powder (15.1 mg, 0.0224 mmol, 79% yield). The materialwas analyzed by ¹H NMR in D₂O and LC-MS.

Example 5 Preparation of β-Alanine Co-Salt of the Hydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (16.7 mg, 0.0285 mmol, 1.0 equiv) wasdissolved in 1.5 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (1.5 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (4.52mg, 0.0314 mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reactionmixture was stirred for 2 h and poured into a separatory funnel with 3.0mL water. The organic layer was extracted and then washed withadditional 2.0 mL water. The combined aqueous layers were lyophilized toyield 4 as a white fluffy powder (12 mg, 0.0237 mmol, 83% yield). Thematerial was analyzed by ¹H NMR in D₂O and LC-MS.

Example 6 Preparation of N-Methyl Glycine Co-Salt of the Hydroquinone of17-AAG

17-Allylaminogeldanamycin (1) (15.1 mg, 0.0258 mmol, 1.0 equiv) wasdissolved in 1.5 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (1.5 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (3.7 mg,0.0258 mmol, 1.0 equiv) in 0.20 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 5 asa white fluffy powder (15.4 mg, 0.0234 mmol, 91% yield). The materialwas analyzed by ¹H NMR in D₂O and LC-MS.

Example 7 Preparation of Piperidine Carboxylate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (16 mg, 0.027 mmol, 1.0 equiv) wasdissolved in 1.5 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (1.5 mL). The deep purple solutionturned yellow attar 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.25 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (5.5 mg,0.03 mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 6 asa white fluffy powder (11.4 mg, 0.019 mmol, 60% yield). The material wasanalyzed by ¹H NMR in D₂O and LC-MS.

Example 8 Preparation of Glycine Co-Salt of the Hydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (16.2 mg, 0.028 mmol, 1.0 equiv) wasdissolved in 1.5 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (1.5 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (3.4 mg,0.03 mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 7 asa white fluffy powder (3.1 mg, 0.0051 mmol, 19% yield, 3:1 mixtures ofphenol regioisomers). The material was analyzed by ¹H NMR in D₂O andLC-MS.

Example 9 Preparation of 2-Amino-2-ethyl-butyrate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (48 mg, 0.082 mmol, 1.0 equiv) wasdissolved in 4.8 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (4.8 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 1 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (16.8mg, 0.09 mmol, 1.1 equiv) in 1 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 8 asa white fluffy powder (24.7 mg, 0.034 mmol, 41% yield). The material wasanalyzed by ¹H NMR in D₂O and LC-MS.

Example 10 Preparation of 1-Amino-Cyclopropanecarboxylate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (48 mg, 0.082 mmol, 1.0 equiv) wasdissolved in 4.8 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (4.8 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 1 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (14.1mg, 0.09 mmol, 1.1 equiv) in 1 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 9 asa white fluffy powder (36.2 mg, 0.051 mmol, 62% yield). The material wasanalyzed by ¹H NMR in D₂O and LC-MS.

Example 11 Preparation of Carboxylate Co-Salt of the Hydroquinone of17-AAG

17-Allylaminogeldanamycin (1) (24 mg, 0.041 mmol, 1.0 equiv) wasdissolved in 2.4 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (2.4 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (7.8 mg,0.045 mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 10as a white fluffy powder (25.8 mg, 0.038 mmol, 92% yield). The materialwas analyzed by ¹H NMR in D₂O and LC-MS.

Example 12 Preparation of 1-Amino-cyclopentanecarboxylate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (48 mg, 0.082 mmol, 1.0 equiv) wasdissolved in 4.8 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (4.8 mL). The deep purple solutionturned yellow after 5 min and the mixture was stirred for an additional25 min. The organic layer was removed via syringe and the aqueoussolution was extracted with an additional 0.30 mL dichloromethane. Thecombined organic solutions were washed with brine (1.0 mL) and thenadded directly to a solution of the acid chloride hydrochloride (17 mg,0.09 mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixturewas stirred for 2 h and poured into a separatory funnel with 3.0 mLwater. The organic layer was extracted and then washed with additional2.0 mL water. The combined aqueous layers were lyophilized to yield 11as a white fluffy powder (34.3 mg, 0.049 mmol, 60% yield). The materialwas analyzed by ¹H NMR in D₂O and LC-MS.

Example 13 Preparation of N-Methyl Piperidinecarboxylate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (21.8 Mg, 0.038 mmol, 1.0 equiv) wasdissolved in 2 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (2 mL). The deep purple solution turnedyellow after 5 min and the mixture was stirred for an additional 25 min.The organic layer was removed via syringe and the aqueous solution wasextracted with an additional 0.30 mL dichloromethane. The combinedorganic solutions were washed with brine (1.0 mL) and then addeddirectly to a solution of the acid chloride hydrochloride (8.1 mg, 0.041mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixture wasstirred for 2 h and poured into a separatory funnel with 3.0 mL water.The organic layer was extracted and then washed with additional 2.0 mLwater. The combined aqueous layers were lyophilized to yield 12 as awhite fluffy powder (15.2 mg, 0.0213 mmol, 56% yield). The material wasanalyzed by ¹H NMR in D₂O and LC-MS.

Example 14 Preparation of N,N,N-Trimethylammonium Acetate Co-Salt of theHydroquinone of 17-AAG

17-Allylaminogeldanamycin (1) (113 mg, 0.19 mmol, 1.0 equiv) wasdissolved in 2 mL dichloromethane and stirred with a 10% aqueoussolution of sodium hydrosulfite (2 mL). The deep purple solution turnedyellow after 5 min and the mixture was stirred for an additional 25 min.The organic layer was removed via syringe and the aqueous solution wasextracted with an additional 0.30 mL dichloromethane. The combinedorganic solutions were washed with brine (1.0 mL) and then addeddirectly to a solution of the acid chloride hydrochloride (33 mg, 0.21mmol, 1.1 equiv) in 0.20 mL dichloromethane. The reaction mixture wasstirred for 2 h and poured into a separatory funnel with 3.0 mL water.The organic layer was extracted and then washed with additional 2.0 mLwater. The combined aqueous layers were lyophilized to yield 13 as awhite fluffy powder (78 mg, 0.11 mmol, 57% yield). The material wasanalyzed by ¹H NMR in CDCl₃/deuterated DMSO (6:1) and LC-MS.

Example 15 Preparation of Air-Stable Hydroquinone Derivatives of 17-AAGfrom Geldanamycin

Geldanamycin (28) (0.14 g, 0.25 mmol, 1.0 equiv) add to a 10 mL vialfollowed by a solution of allyl amine (0.075 mL, 1.0 mmol, 4 equiv.) inMeTHF (0.625 mL). The resulting slurry is heated to 40° C. undernitrogen for 10 hours. The reaction mixture was then cooled to roomtemperature, diluted with 1.0 mL of MeTHF, washed with a saturated NH₄Clsolution (1.5 mL) and saturated NaCl (1.5 mL). The organic layer wasthen collected and treated with a freshly prepared aqueous solution ofsodium hydrosulfite (1 mL, 20% (m/m)) with vigorous stirring undernitrogen for 45 minutes. The aqueous layer was then removed and theorganic layer was then washed with 1.5 mL of degassed water. The organicsolution was then dried by azetropic removal of water using MeTHF. Thiswas accomplished by the addition of 2 mL of MeTHF and then concentration(about 2 mL) of the resulting solution under reduced pressure at 70° C.The resulting solution is then cooled to 0° C. in an ice bath and thenα-aminoisobutyric acid chloride hydrochloride (0.04 g, 0.25 mmol, 1.0equiv.) is added under nitrogen. The reaction mixture is stirred for 3hours at which point the solid is collected by filtration and washedwith MeTHF (2×2 mL). The solid is then dried under reduced pressure toyield the product as a yellow powder (171 mg, 0.2425 mmol, 97% overallyield).

Example 16 Crystallization of Hydroquinone Co-Salt Forms of 17-AAG

Compound 7 is dissolved in the minimal amount of MeOH and then EtOAc isslowly added drop wise until the turbidity persists. The mixture is thenallowed to stand for 14 hours and then the solid is collected byfiltration, washed with EtOAc and dried under reduced pressure.

Example 17 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.450 g, 0.768 mmol, 1.0 equiv) is dissolved indichloromethane (50 mL) and stirred with a 10% aqueous solution ofsodium hydrosulfite (50 mL). The solution is stirred for 30 minutes. Theorganic layer was collected, dried over Na₂SO₄, filtered and transferredto a round bottom flask. To this solution was added a solution of HCl indioxane (4 N, 0.211 mL, 1.1 equiv.). The resulting mixture was allowedto stir under nitrogen for 30 minutes. A yellow solid slowly crashed outof solution. The yellow solid was purified by recrystallization formMeOH/EtOAc to yield 0.386 g of the IPI-504 (15).

Example 18 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.30 g, 0.5 mmol, 1.0 equiv) is dissolved inMTBE (3 mL) and stirred with a 20% aqueous solution of sodiumhydrosulfite (2 mL). The solution is stirred for 60 minutes. The organiclayer was collected, washed with brine, and transferred to a roundbottom flask. This solution was cooled −5° C. and put under nitrogen. Tothis solution was added a solution of H₂SO₄ in denatured ethanol (0.50mmol of H₂SO₄ in 0.5 mL of EtOH) dropwise. The resulting mixture wasallowed to stir under nitrogen and warm to RT. The yellow slurry wasstirred for an additional 30 minutes at RT and then was concentrated.MTBE (7 mL) was added and the suspension was filtered. The yellow solidthat was collected was washed with MTBE and dried under reduced pressureto yield 0.30 g of the desired product.

Example 19 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.30 g, 0.5 mmol, 1.0 equiv) is dissolved inDCM (6 mL) and stirred with a 10% aqueous solution of sodiumhydrosulfite (3.5 mL). The solution is stirred for 60 minutes. Theorganic layer was collected, washed with brine, and 1.2 mL (calc 0.1mmol of hydroquinone) transferred to a round bottom flask. This solutionwas put under nitrogen. To this solution was added a solution ofp-toluenesolfonic acid in denatured IPA (0.100 mmol of p-toluenesolfonicin 0.25 mL of IPA) dropwise. The resulting mixture was allowed to stirunder nitrogen for 1 hour, at which point the mixture was concentratedand the crude mass was reslurried from EtOAc/MTBE. The solid wascollected by filtration and dried under reduced pressure to yield 0.068g of the desired product.

Example 20 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.30 g, 0.5 mmol, 1.0 equiv) is dissolved inDCM (6 mL) and stirred with a 10% aqueous solution of sodiumhydrosulfite (3.5 mL). The solution is stirred for 60 minutes. Theorganic layer was collected, washed with brine, and 1.2 mL (calc 0.1mmol of hydroquinone) transferred to a round bottom flask. This solutionwas put under nitrogen. To this solution was added a solution ofd-camphorsulonic acid in denatured IPA (0.100 mmol of d-camphorsulonicacid in 0.25 mL of IPA) dropwise. The resulting mixture was allowed tostir under nitrogen for 1 hour, at which point the mixture wasconcentrated and the crude mass was reslurried from EtOAc/MTBE. Thesolid was collected by filtration and dried under reduced pressure toyield 0.051 g of the desired product.

Example 21 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.30 g, 0.5 mmol, 1.0 equiv) is dissolved inDCM (6 mL) and stirred with a 10% aqueous solution of sodiumhydrosulfite (3.5 mL). The solution is stirred for 60 minutes. Theorganic layer was collected, washed with brine, and 1.2 mL (calc 0.1mmol of hydroquinone) transferred to a round bottom flask. This solutionwas put under nitrogen. To this solution was added a solution of H₃PO₄in denatured IPA (0.100 mmol of H₃PO₄ in 0.25 mL of IPA) dropwise. Theresulting mixture was allowed to stir under nitrogen for 1 hour, atwhich point the mixture was concentrated and the crude mass wasreslurried from EtOAc/MTBE. The solid was collected by filtration anddried under reduced pressure to yield 0.050 g of the desired product.

Example 22 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.50 g, 0.8 mmol, 1.0 equiv) is dissolved inDCM (8 mL) and stirred with a 15% aqueous solution of sodiumhydrosulfite (4 mL). The solution is stirred for 60 minutes. The organiclayer was collected, washed with brine, and 2 mL (calc 0.2 mmol ofhydroquinone) transferred to a round bottom flask. This solution was putunder nitrogen. To this solution was added a solution of MeSO₃H indenatured IPA (0.200 mmol of MeSO₃H in 0.4 mL of IPA) dropwise. Theresulting mixture was allowed to stir under nitrogen for 1 hour, atwhich point the mixture was concentrated and the crude mass wasreslurried from EtOAc. The solid was collected by filtration and driedunder reduced pressure to yield 0.112 g of the desired product.

Example 23 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

Compound of Formula (1) (0.50 g, 0.8 mmol, 1.0 equiv) is dissolved inDCM (8 mL) and stirred with a 15% aqueous solution of sodiumhydrosulfite (4 mL). The solution is stirred for 60 minutes. The organiclayer was collected, washed with brine, and 2 mL (calc 0.2 mmol ofhydroquinone) transferred to a round bottom flask. This solution was putunder nitrogen. To this solution was added a solution of PhSO₃H indenatured IPA (0.200 mmol of PhSO₃H in 0.4 mL of IPA) dropwise. Theresulting mixture was allowed to stir under nitrogen for 1 hour, atwhich point the mixture was concentrated and the crude mass wasreslurried from EtOAc. The solid was collected by filtration and driedunder reduced pressure to yield 0.118 g of the desired product.

Example 24 Preparation of Air-Stable Hydroquinone Derivatives of theGeldanamycin Family of Molecules

17-Allylamino-17-Demethoxygeldanamycin (10.0 g, 17.1 mmol) in ethylacetate (200 mL) was stirred vigorously with a freshly prepared solutionof 10% aqueous sodium hydrosulfite (200 mL) for 2 h at ambienttemperature. The color changed from dark purple to bright yellow,indicating a complete reaction. The layers were separated and theorganic phase was dried with magnesium sulfate (15 g). The drying agentwas rinsed with ethyl acetate (50 mL). The combined filtrate wasacidified with 1.5 M hydrogen chloride in ethyl acetate (12 mL) to pH 2over 20 min. The resulting slurry was stirred for 1.5 h at ambienttemperature. The solids were isolated by filtration, rinsed with ethylacetate (50 mL) and dried at 40° C., 1 mm Hg, for 16 h to afford 9.9 g(91%) of off-white solid. Crude hydroquinone hydrochloride (2.5 g) wasadded to a stirred solution of 5% 0.01 N aq. hydrochloric acid inmethanol (5 mL). The resulting solution was clarified by filtration thendiluted with acetone (70 mL). Solids appeared after 2-3 min. Theresulting slurry was stirred for 3 h at ambient temperature, then for 1h at 0-5° C. The solids were isolated by filtration, rinsed with acetone(15 mL) and dried

Example 25

17-Allylamino-17-Demethoxygeldanamycin (0.350 g, 0.598 mmol) in ethylacetate (7 mL) was stirred vigorously with a freshly prepared solutionof 10% aqueous sodium hydrosulfite (7 mL) for 1 h at ambienttemperature. The color changed from dark purple to bright yellow,indicating a complete reaction. The layers were separated and theorganic phase was dried with magnesium sulfate (1 g). The drying agentwas rinsed with ethyl acetate (1 mL). The combined organic layers werestirred at room temperature and to it was added triphosgene (0.079 g,0.239 mmol). A precipitate formed and the resulting mixture was allowedto stir for 2 hr. At which point the solid was filtered off and theorganic solution was concentrated. The crude product was purified bycolumn chromatography to yield 17 mg of the desired product.

Example 26

17-Allylamino-17-Demethoxygeldanamycin (0.825 g, 0.141 mmol) in ethylacetate (17.5 mL) was stirred vigorously with a freshly preparedsolution of 10% aqueous sodium hydrosulfite (17.5 mL) for 1 h at ambienttemperature. The color changed from dark purple to bright yellow,indicating a complete reaction. The layers were separated and theorganic phase was dried with magnesium sulfate (1 g). The drying agentwas rinsed with ethyl acetate (1 mL). The combined organic layers werestirred at room temperature and to it was added bromoacetyl chloride(0.222 g, 1.41 mmol). A precipitate formed and the resulting mixture wasallowed to stir for 12 hr. At which point the solid was filtered off andthe organic solution was concentrated. The crude material was dissolvedin a 1:1 mixture of THF/Water (16 mL). Na₂CO₃ (10 equiv) was added andthe resulting mixture was vigorously shaken for 1 hr. The reaction wasquenched with saturated NaHCO₃, washed with brine, dried over MgSO₄ andconcentrated to yield 1.1 mg of the desired product.

Example 27

Geldanamycin (1.12 g, 2 mmol, 1 equiv) was added to anhydrous DCM (5mL). NH₃ in MeOH was added to this solution (9 mL, 100 mmol, 50 equiv)and was allowed to stir for 24 hours. At which point the reactionsolution was diluted with DCM and extracted with water, followed bydilute HCl. The organic layer was collected washed with brine, driedover Na₂SO₄ and concentrated to yield a purple solid. This solid wasrecrystallized twice from acetone/heptanes to yield 0.239 of17-amino-17-demethoxygeldanamycin.

17-amino-17-demethoxygeldanamycin (0.55 g, 1 mmol, 1 equiv) wasdissolved in EtOAc (100 mL). A freshly prepared solution of 10% aqueoussodium hydrosulfite (10 mL) was added and stirred for 1 h at ambienttemperature. The color changed from dark purple to bright yellow,indicating a complete reaction. The layers were separated and theorganic phase was dried with magnesium sulfate. The drying agent wasrinsed with ethyl acetate (2×10 mL). The combined filtrate was acidifiedwith 1.5 M hydrogen chloride in ethyl acetate (1 mL) to pH 2 over 20min. The resulting slurry was stirred for 1.5 h at ambient temperature.The solids were isolated by filtration, rinsed with ethyl acetate (10mL) and dried under vacuum to yield the product (0.524 g, 87% yield).

Example 28

Geldanamycin (0.500 g, 0.892 mmol, 1 equiv) was dissolved in THF (10 mL)3-amino-1,2-propanediol (0.813 g, 8.92 mmol, 10 equiv). The reaction wasstirred for 64 hours. The reaction was then quenched with dilute HCl andextracted with EtOAc. The organic layer was collected dried over MgSO4and concentrated under reduced pressure. The crude material was purifiedusing column chromatography to yield 27 mg of the 17-amino substitutedgeldenamycin.

The 17-amino geldanamycin (0.200 g, 0.323 mmol, 1 equiv) was dissolvedin EtOAc (4 mL) and treated with a freshly prepared 10% solution ofNa₂S₂O₄ in water (4 mL). This mixture was vigorously stirred for 1 hour.The organic layer was then collected. The aqueous layer was extractedwith 2×5 mL of EtOAc. The organic layers were combined, washed withwater, dried over Na₂SO₄. The organic layer was then treated with HCl inEtOAc (1.6 M, 0.6 mL) and stirred for 20 minutes. The reaction solutionwas concentrated under reduced pressure to yield the product (0.009 g).

Example 29

Geldanamycin (0.022 g, 0.04 mmol, 1.5 equiv) and BODIPY-FL-EDA-HCl(0.010 g, 0.026 mmol, 1 equiv) were added to anhydrous DCM (2 mL). DIPEA(30 uL, 0.16 mmol, 6 equiv) was added and the reaction solution wasstirred under nitrogen for 72 hours. The reaction was then diluted withDCM, extracted with water, dried over Na₂SO₄ and concentrated underreduced pressure. The crude was purified by column chromatography toyield the 17-amino substituted benzoquinone. This material was dissolvedin EtOAc (20 mL) and treated with a freshly prepared 10% solution ofNa₂S₂O₄ in water (5 mL). This mixture was vigorously stirred for 1 hour.The organic layer was then collected. The aqueous layer was extractedwith 2×5 mL of EtOAc. The organic layers were combined, washed withwater, dried over Na₂SO₄. The organic layer was then treated with HCl inEtOAc (1.6 M, 0.6 mL) and stirred for 20 minutes. The reaction solutionwas then concentrated to dryness under reduce pressure. The crude waspurified by reslurrying the material from EtOAc/MTBE. The solid waswashed with MTBE and dried under reduced pressure to yield the product(0.04 g).

Example 30

Anhydrous ethyl acetate (170 mL) was added to a flask followed by 17-AAG(8.41 g, 1.44 mmol, 1 equiv). The resultant purple mixture was stirredvigorously under nitrogen. A freshly prepared solution of 10%Na₂S₂O₄(aq) (1.682 g in 170 mL of deionized water, 10.1 mmol, 7 equiv)was added and the mixture stirred vigorously for 70 min. The colorchanged from purple to orange indicating a complete reaction. The layerswere allowed to separate and the bottom aqueous layer was removed usinga separatory funnel. The organic layer was dried with MgSO₄. The dryingagent was removed by filtration. The filtrate was transferred to arotary evaporator flask. Ethyl acetate (50 mL) was used, in portions, towash the MgSO₄ pad and the wash filtrate was also added to the rotaryevaporator flask.

The orange-brown mixture was concentrated on the rotary evaporator to anoil. The remaining ethyl acetate was removed under vacuum.

While this mixture was concentrated, a 5.3 M solution of HCl in ethylacetate was prepared. Ethyl acetate (16.8 mL) was added to an Erlenmeyerflask and HCl gas bubbled into the stirring mixture for 1 h (withcooling, acetone/wet ice) to achieve saturation. The solution was thenwarmed to room temperature under a head space of nitrogen.

The oil was dissolved in acetone (252 mL) and transferred to a reactionflask equipped with an addition funnel, a stirrer, a thermometer, and anitrogen atmosphere. The combined filtrate and rinse were acidified over5 min to a final pH of 2.5. The resulting slurry was stirred for 18 minat ambient temperature and the solids were then isolated by filtrationand washed twice with acetone (84 mL). The solid was then dried underreduced pressure to yield the product

Example 31

17-Allylamino-17-Demethoxygeldanamycin (1.0 g, 1.71 mmol) in ethylacetate (20 mL) was stirred vigorously with a freshly prepared solutionof 10% aqueous sodium hydrosulfite (2 g in 20 mL water) for 30 minutesat ambient temperature. The color changed from dark purple to brightyellow, indicating a complete reaction. The layers were separated andthe organic phase was dried with magnesium sulfate (1 g). The reactionsolvent was collected and the drying agent was rinsed with ethyl acetate(1 mL). The combined filtrate was cooled to 0 C and acidified with 1.5 Mhydrogen bromide in ethyl acetate until a precipitate formed. Theresulting slurry was stirred for 30 minutes at ambient temperature. Thesolids were isolated by filtration, rinsed with ethyl acetate (1 mL) anddried at 40° C., 1 mm Hg, for 16 h to afford 0.352 g (31%) of off-whitesolid.

Example 32 Preparation of 50 mM Citrate, 50 mM Ascorbate, pH 3.1, 2.44mM EDTA As the Formulation Buffer for Compounds of the Present Invention

An Example of Formulation Preparation:

For an 1 L preparation of formulation buffer, 9.6 g citric acid (USP),8.8 g ascorbic acid (USP) and 1.0 g EDTA (Ethylenediamine-tetraaceticacid, disodium-calcium salt, dihydrate, USP), was added with ateflon-coated magnetic stir-bar to a 1 L volumetric flask. Sterile waterfor injection (USP) was added to 90-95% of the final volume of theflask. The solution was vigorously stirred to dissolve all solids. ThepH of the buffer was adjusted to 3.1 using a NaOH solution. WFI wasadded to the final volume. The buffer was vacuum filtered through a 0.2micron filter unit. Prior to use, the solution was sparged with nitrogenfor 1-2 h. The formulation buffer was stored under nitrogen at 4° C. ina closed container.

Formulated Drug Product Preparation:

The drug product was formulated at 4° C. by controlled dissolution ofthe solid compound 15 with pre-chilled nitrogen-sparged formulationbuffer in a water-cooled jacketed vessel under a nitrogen headspace.Formulated compound 15 solution stored at 4° C. wider a nitrogenheadspace.

An Example of the Formulated Drug Product Preparation in Solution isGiven Below.

A 10 mL volumetric flask was charged with solid compound 15 (500 mg) andpurged with nitrogen. Formulation buffer (50 mM citrate, 50 mMascorbate, 2.44 mM EDTA, pH 3.1) was sparged with nitrogen untildissolved oxygen content is <0.5 mg/L and chilled on ice. A portion ofthe buffer (approximately 5-7 mL) was added to the volumetric flask andvigorously shaken until all solid was dissolved. Buffer was then addedto the 10 mL mark on the volumetric flask. The solution was kept cold onice as much as possible. A 10 mL syringe with syringe filter (Millipore,Durapore membrane, 0.2 micron) was used to filter the clear, slightlytan solution into a glass vial (USP Type I). The formulated compound 15solution stored at 4° C. under a nitrogen headspace.

An Example of the Formulated Drug Product Preparation in Solid Form isGiven Below.

52.50 g of sterile water was added to a 100 mL flask equipped with amagnetic stir bar. 6.305 g of citric acid monohydrate was added to the100 mL flask and the resulting mixture was stirred until all of thecitric acid dissolved into solution. 5.284 g of L-ascorbic acid was thenadded to the 100 mL flask and the solution was stirred until all of theascorbic acid dissolved into solution. 0.600 g of edetate calciumdisodium was then added to the 100 mL flask and resulting mixture wasstirred until all of the edetate calcium disodium had dissolved intosolution. The pH of the solution was then adjusted to a pH of 3.1 byslowly adding a 5 M sodium hydroxide solution in water. The solution wasthen sparged with filtered (Millipak 20, 0.22 micron durapore) nitrogenfor 2 hours. 52.04 g of the sparged solution was then cooled to 0° C.under nitrogen with stirring. 2.80 g of compound 15 was added and theresulting mixture was stirred until all of compound 15 was dissolved.This solution was sterile filtered using a 0.22 micron pore-sizeDurapore Millipak 200 filter at 0° C. The headspace of the receivingvessel was then flushed with filtered (Millipak 20, 0.22 microndurapore) nitrogen.

The receiving vessel was then placed in a lyophilizer, which had beenpre-cooled to −40° C. The lyophilizer chamber was held at −40° C. for 3hours at 1 atm. The pressure of the lyophilizer chamber was then rampedto 100 micron over one hour. Then the temperature of the chamber wasramped to −20° C. over 2 hour and the vacuum was held at 100 micron. Thetemperature of the chamber was then ramped to 0° C. over 2 hours and thevacuum was held at 100 micron. Then the temperature of the chamber wasramped to 0° C. over 2 hours and the vacuum was held at 100 micron. Thetemperature of the chamber was then ramped to +10° C. over 2 hours andthe vacuum was held at 100 micron. Then the temperature of the chamberwas ramped to +20° C. over 2 hours and the vacuum was held at 100micron. The temperature of the chamber was then maintained at +20° C.for 48 hours and the vacuum was held at 100 micron. The chamber was thenpurged with nitrogen and a stopper was attached to the vessel containingthe formulation. The formulation was stored at −20° C.

Example 33 Preparation of 75 mM Citrate, 170 mM Ascorbate, pH 3.0, 2.44mM EDTA, 1% (w/v) Gamma-Cyclodextrin as the Formulation Buffer forCompounds of the Present Invention

An Example of Formulation Preparation:

For a 1 L preparation of formulation buffer, 14.4 g citric acid, 30 g ofascorbic acid, 10 g of gamma-cyclodextrin (cyclooctaamylose) and 1.0 gEDTA was added with a teflon-coated magnetic stir-bar to a 1 Lvolumetric flask. Sterile water for injection was added to 90-95% of thefinal volume of the flask. The solution was vigorously stirred todissolve all solids. The pH of the buffer was adjusted to 3.0 using aNaOH solution (NF grade). WFI was added to the final volume. The bufferwas vacuum filtered through a 0.2 micron filter unit. Prior to use, thesolution was sparged with nitrogen for 1-2 h. The formulation buffer wasstored under nitrogen at 4° C. in a closed container.

Formulated Drug Product Preparation:

The drug product was formulated at 4° C. by controlled dissolution ofthe solid compound 15 with pre-chilled nitrogen-sparged formulationbuffer under a nitrogen headspace. Formulated compound 15 solution wasstored at 4° C. under a nitrogen headspace.

Example 34 Preparation of 50 mM citrate, 25 mM Ascorbate, 1% (v/v)Polysorbate-80, 0.1% (w/v) EDTA, pH 3.0 as the Formulation Buffer forCompounds of the Present Invention

An Example of Formulation Preparation:

For a 1 L preparation of formulation buffer, 9.6 g citric acid, 4.4 g ofascorbic acid, 10 mL of polysorbate-80 and 1.0 g EDTA(Ethylenediamine-tetraacetic acid, disodium-calcium salt, dihydrate) wasadded with a teflon-coated magnetic stir-bar to a 1 L volumetric flask.Sterile water for injection (WFI) was added to 90-95% of the finalvolume of the flask. The solution was vigorously stirred to dissolve allsolids. The pH of the buffer was adjusted to 3.0 using a NaOH solution.WFI was added to the final volume. The buffer was vacuum filteredthrough a 0.2 micron filter unit. Prior to use, the solution was spargedwith nitrogen for 1-2 h. The formulation buffer was stored undernitrogen at 4° C. in a closed container.

Formulated Drug Product Preparation:

The drug product was formulated by controlled dissolution of the solidcompound 15 with nitrogen-sparged formulation buffer. Formulatedcompound 15 solution stored at 4° C. under a nitrogen headspace.

Example 35

Materials and Methods for In Vitro Analysis

Cell Cultures

The human cancer cell lines SKBr3, MV4-11, K562, SK-MEL-28, LnCAP, andMDA-MB-468 were obtained from the American Type Culture Collection(Manassas, Va.). The multiple myeloma RPMI-8226 and MM1.s cells werefrom Dr. Teru Hideshima (Jerome Lipper Multiple Myeloma Center, DanaFarber Cancer Institute, Boston, Mass., USA.). All the cell lines weredetermined to be mycoplasma-free. The cells were maintained in RPMI-1640medium supplemented with 10% heat-inactivated FBS, 50 units/mLstreptomycin and 50 units/mL penicillin, and incubated at 37° C. in 5%CO₂. Adherent cells were dissociated with 0.05% trypsin and 0.02% EDTAin phosphate buffer saline (PBS) without calcium and magnesium prior toplating for experimentation.

In Vitro Analysis

MM1.s Cell Cytotoxicity

Alamar Blue assay. MM1.s cells (50,000/well) were incubated for 72 hwith increasing concentrations of the test compound. Alamar blue wasadded to the wells and fluorescence measured 4 h after incubation at 37°C.

SKBr3 Cell Cytotoxicity

SKBr3 Cells were incubated for 72 h with increasing concentrations ofthe test compound. For the viability studies Alamar blue was added andwells read after a 6 h incubation.

MDA-MB-468 Cell Cytotoxicity

MDA-MB-468 Cells were incubated for 72 h with increasing concentrationsof the test compound. For the viability studies Alamar blue was addedand wells read after 6 h of incubation.

MV4-11 Cell Cytotoxicity

MV4-11 cells were incubated for 3 days with increasing concentrations ofthe test compound. Cell viability was assessed using an Alamar blue readout.

K562 Cell Cytotoxicity

K562 cells were incubated with increasing concentrations of the testcompound. Cell viability was assessed using an Alamar blue read out

SK-MEL-28 Cell Cytotoxicity

Increasing concentrations of the test compound was added to SK-MEL-28cells in culture for 2, 3 or 4 days and the viability of the cells wasmeasured using Alamar blue.

LnCAP Cell Cytotoxicity

Increasing concentrations of the test compound added to LnCAP cells inculture for 4 days and the viability of the cells was measured usingAlamar blue.

Example 36 Competitive Binding to HSP90 Assay for 17-AAG and Compound 15

Materials

Native human Hsp90 protein isolated from HeLa cells (SPP-770),recombinant canine Grp94 (SPP-766), and recombinant human Hsp70(ESP-555) were purchased from Stressgen Biotechnologies (Victoria, BC).Complete™ protease inhibitor tablets were obtained from RocheDiagnostics (Indianapolis, Ind.). All other chemicals and reagents werepurchased from Sigma-Aldrich and are analytical grade or higher.

FP Binding Assay—Binding of BODIPY-GDM to Purified Proteins

The procedures were modified based on Llauger-Bufi et al. (Llauger-BuffL, Felts S J, Huezo H, Rosen N, Chiosis G. Synthesis of novelfluorescent probes for the molecular chaperone Hsp90. Bioorg Med ChemLett (2003) 13:3975-3978) and Kim et al. (Kim J, Felts S, Llauger L, HeH, Huezo H, Rosen N, Chiosis G. Development of a fluorescencepolarization assay for the molecular chaperone Hsp90. J Biomol Screening(2004) 9:375-381). A 20 nM BODIPY-GDM solution was freshly prepared inFP binding assay buffer [20 mM HEPES-KOH, pH 7.3, 1.0 mM EDTA, 100 mMpotassium chloride, 5.0 mM magnesium chloride, 0.01% NP-40, 0.1 mg/mLBovine γ-globulin (BGG), 1.0 mM DTT, and Complete™ protease inhibitor]from a 20 μM stock solution in DMSO. Ten microliters of this solutionwere dispensed into each well of a black round-bottom 384-wellmicroplate (Corning #3676). An equal volume of serially diluted humanHsp90 solution in FP binding assay buffer was then added to give finalconcentrations of 10 nM BODIPY-GDM and Hsp90 varying in concentrationfrom 6.25 μM to 0.10 nM. The final DMSO concentration was 0.05%. After3-h incubation at 30° C., fluorescence anisotropy was measured on anEnVision 2100 multilabel plate reader equipped with a 485 nm excitationfilter and a 535 nm P/S emission filter (Perkin Elmer, Boston, Mass.).

Competition by 17-AAG and Analogues

17-AAG and compound 15 were first dissolved in DMSO to give stocksolutions at 5.0 and 1.0 mM concentrations. A dilution series for eachcompound was freshly prepared in FP binding assay buffer from 20 μM to0.20 nM. A solution containing 20 nM BODIPY-GDM and 80 nM Hsp90 was alsoprepared in FP binding assay buffer (0.10% DMSO). In a 384-wellmicroplate, 10 μL of the solution containing BODIPY-GDM and Hsp90 wasmixed with an equal volume of the compound dilution series to give finalconcentrations of 10 nM BODIPY-GDM, 40 nM Hsp90, and varyingconcentrations of the specific compound from 10 μM to 0.10 nM. Themaximal DMSO concentration is 0.25% in the final assay mixture. After3-h incubation at 30° C., fluorescence anisotropy was measured on anEnVision 2100 plate reader.

Assays were performed under nitrogen atmosphere in a LabMaster glove box(M. Braun, Stratham, N H). Typically, 50 mL of FP binding assay bufferwas deoxygenated by repeated cycles of evacuation and flushing withargon. Protein solutions and compound stock solutions in DMSO werebrought into the glove box as frozen-liquids. All dilutions andsubsequent mixing of assay components were performed inside the glovebox as described above. After 3-h incubation at 30° C., the microplatewas brought out of the glove box, and fluorescence anisotropy wasimmediately measured on an EnVision 2100 plate reader.

Data Analysis

Binding of BODIPY-GDM to Hsp90 results in simultaneous increases influorescence anisotropy (FA) and intensity (FI). To calculate K_(d), abinding curve of FI versus Hsp90 (monomer) concentration is fitted by afour-parameter logistic function:

${FI} = {{FI}_{\min} + \frac{\left( {{FI}_{\max} - {FI}_{\min}} \right)}{1 + \left( {{EC}_{50}/\lbrack E\rbrack_{total}} \right)^{Hill}}}$with Hill coefficient forced to 1 for simplicity. From the values ofFI_(max) (bound ligand) and FI_(min) (free ligand), a Q factor iscalculated by:

$Q = \frac{{FI}_{\max}}{{FI}_{\min}}$The binding curve of FA vs. Hsp90 concentration is subsequently fittedusing the program SCIENTIST and the following equations:

$\begin{matrix}{K_{d} = \frac{\lbrack E\rbrack_{free} \times \lbrack L\rbrack_{free}}{\lbrack{EL}\rbrack}} \\{= \frac{\left( {\lbrack E\rbrack_{total} - \lbrack{EL}\rbrack} \right) \times \left( {\lbrack L\rbrack_{total} - \lbrack{EL}\rbrack} \right)}{\lbrack{EL}\rbrack}}\end{matrix}$and FA being expressed as the weighted sum of contributions from thefree and bound forms of ligand:

$\begin{matrix}{{FA} = \frac{{FA}_{\min} \times \lbrack L\rbrack_{free} \times {FA}_{\max} \times Q \times \lbrack{EL}\rbrack}{\lbrack L\rbrack_{free} + {Q \times \lbrack{EL}\rbrack}}} \\{= \frac{{{FA}_{\min} \times \left( {\lbrack L\rbrack_{total} - \lbrack{EL}\rbrack} \right)} + {{FA}_{\max} \times Q \times \lbrack{EL}\rbrack}}{\left( {\lbrack L\rbrack_{total} - \lbrack{EL}\rbrack} \right) + {Q \times \lbrack{EL}\rbrack}}}\end{matrix}$Competition binding curves are analyzed in a similar fashion. Thedecrease in FI as a function of increasing inhibitor concentration isdescribed by the logistic function:

${FI} = {{FI}_{\min} + \frac{\left( {{FI}_{\max} - {FI}_{\min}} \right)}{1 + \left( {\lbrack I\rbrack_{total}/{EC}_{50}} \right)^{Hill}}}$The curve of FA vs. inhibitor concentration is subsequently fitted usingimplicit function for competitive binding equilibrium to give K_(i):

$\begin{matrix}{\lbrack E\rbrack_{total} = {\lbrack E\rbrack_{free} + \lbrack{EL}\rbrack + \lbrack{EI}\rbrack}} \\{= {\lbrack E\rbrack_{free} + \frac{\lbrack E\rbrack_{free} \times \lbrack L\rbrack_{total}}{\lbrack E\rbrack_{free} + K_{d}} + \frac{\lbrack E\rbrack_{free} \times \lbrack I\rbrack_{total}}{\lbrack E\rbrack_{free} + K_{i}}}}\end{matrix}$given the known values of [E]_(total), [L]_(total), and K_(d).Summary

This experiment demonstrated that both quinone and hydroquinoneansamycins (e.g., 17-AAG and compound 15) are active HSP90 inhibitors.

Example 37

In Vivo Analysis

Multiple Myeloma Model

The effects of the test compound were studied in a human multiplemyeloma cell line RPMI-8226 in male SCID/NOD mice. In this study, malemice were implanted subcutaneously with RPMI-8226 cells (1×10⁷ cells).When the average tumor size reached 100 mm³, animals were randomlyassigned to treatment groups (N=10-15/group) to receive either vehicle(50 mM citrate, 50 mM ascorbate, 2.4 mM EDTA adjusted to pH 3.0) or 100mg/kg (300 mg/m²) of the test compound three consecutive days per week.The test article or vehicle was administered intravenously (IV) via thetail vein in a volume of 0.2 mL over approximately 20 seconds (sec). Theanimals were sacrificed after 45 days and tumor volumes compared.

Breast Carcinoma Model

A study was performed in the MDA-MB-468 breast carcinoma model to assessthe ability of the test compound to reduce subcutaneous tumor burden. Inthis study, female nu/nu athymic mice were implanted subcutaneously withMDA-MB-468 cells (1×10⁷ cells). When the average tumor size reached 100mm³, animals were randomly assigned (N=10-15/group) to one of thefollowing treatment groups; vehicle or the test compound at 100 mg/kg(300 mg/m²) twice weekly every week. The test article or vehicle wasadministered intravenously (IV) via the tail vein in a volume of 0.2 mLover approximately 20 seconds (sec). The animals were sacrificed after120 days and tumor volumes compared.

Ovarian Carcinoma Model

A study was performed in the SKOV-3 ovarian mouse xenograft model toassess the ability of the test compound to reduce subcutaneous tumorburden. In this study, female nu/nu athymic mice were implantedsubcutaneously with SKOV-3 cells (1×10⁷ cells). When the average tumorsize reached 100 mm³, animals were randomly assigned to treatment groups(N=10-15/group) to receive either vehicle, the test compound at 100mg/kg (300 mg/m²) twice weekly. The test article or vehicle wasadministered intravenously (IV) via the tail vein in a volume of 0.1 mLover approximately 10 seconds (sec). The animals were sacrificed after88 days and tumor volumes compared.

Murine Lewis Lung Model

A study was performed in the mouse Lewis lung model to assess theability of the compounds of the present invention to reduce bothsubcutaneous tumor burden as well as the incidence of lung metastasis.In this study C57B1/6 mice were implanted subcutaneously with Lewis lungcells (1×10⁶ cells). When the average tumor size reached 71 mm³ animals(N=10-15/group) were randomly assigned to the following treatmentgroups: vehicle and compound 15 75 mg/m² Monday, Wednesday and Friday(MWF) for 3 cycles. Each cycle consisted of 5 days per week oftreatment. The test article or vehicle was administered via the tailvein in a volume of 0.2 mL over approximately 30 sec. The animals weresacrificed after 25 days and tumor volumes were compared.

Prostate Carcinoma

Two studies were performed in mouse PC-3 prostate xenograft models toassess the ability of the test compound to reduce subcutaneous tumorburden as a single agent or in combination with current standard ofcare. In both studies male nu/nu athymic mice were implantedsubcutaneously with PC-3 cells (1×10⁷ cells). When the average tumorsize reached 100 mm³ animals were randomly assigned to treatment groups(N=10-15/group). In the first study mice received either vehicle, thetest compound 100 mg/kg (300 mg/m²) twice weekly. The test article orvehicle was administered via the tail vein in a volume of 0.2 mL overapproximately 20 sec. The animals were sacrificed after 64 days andtumor volumes compared.

A second study was performed in this model to assess the test compoundin combination with the standard of care, Taxotere. In this studyseparate groups of 10-15 mice each were randomly assigned to receivevehicle, the test compound 100 mg/kg (300 mg/m²) twice weekly, Taxotere5 mg/kg (15 mg/m²) once weekly or combination of the test compound withTaxotere. The animals were sacrificed after 64 days and tumor volumescompared.

Example 38

Biological Results

The results from the biological activity analysis of the hydroquinonesof the invention are presented below. All values are expressed as themean±SEM. Data analysis consisted of a one way analysis of variance andif appropriate followed by Dunnets test to assess differences betweenvehicle and treatment groups. Differences are considered significant atp<0.05.

In Vitro Results

Cell Line Compound 15 (EC₅₀) 17-AAG (EC₅₀) MM1.s 307 nM 306 nM SKBr3 32nM 34 nM MDA-MB-468 335 nM 356 nM MV4-11 25 nM 38 nM K562 29 nM 50 nMSK-MEL-28 200 nM — LnCAP 73 nM —In Vivo Results

% Tumor Growth Compared to Vehicle Cell Line Compound 15 Compound 15 +Taxotere RPMI-8226 71% — MDA-MB-468 76% — SKOV-3 59% — Lewis Lung Cell60% — PC-3 50% 84%Binding of Compound 15 and 17-AAG to HSP90

Compound K_(i) Compound 15 28 nM 17-AAG 67 nM

EQUIVALENTS & INCORPORATION BY REFERENCE

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areincluded within the spirit and purview of this application and scope ofthe appended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

1. A pharmaceutical composition, comprising a pharmaceuticallyacceptable excipient; and a compound of the formula:

wherein independently for each occurrence: W is oxygen or sulfur; Q isoxygen, NR, N(acyl) or a bond; R for each occurrence is independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl; R₁ ishydroxyl, alkoxyl, —OC(O)R₈, —OC(O)OR₉, —OC(O)NR₁₀R₁₁, —OSO₂R₁₂,—OC(O)NHSO₂NR₁₃R₁₄, —NR₁₃R₁₄, or halide; and R₂ is hydrogen, alkyl, oraralkyl; or R₁ and R₂ taken together, along with the carbon to whichthey are bonded, represent —(C═O)—, —(C═N—OR)—, —(C═N—NHR)—, or—(C═N—R)—; R₃ and R₄ are each independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, and—[(CR₂)_(p)]—R₁₆; or R₃ taken together with R₄ represent a 4-8 memberedoptionally substituted heterocyclic ring; R₅ is selected from the groupconsisting of H, alkyl, aralkyl, and a group having the formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ are both hydrogen; or R₆ and R₇ takentogether form a bond; R₈ is hydrogen, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆; R₉ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆; R₁₀ and R₁₁ are each independently selected from thegroup consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, and—[(CR₂)_(p)]—R₁₆; or R₁₀ and R₁₁ taken together with the nitrogen towhich they are bonded represent a 4-8 membered optionally substitutedheterocyclic ring; R₁₂ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆; R₁₃ and R₁₄ are each independently selected from thegroup consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, and—[(CR₂)_(p)]—R₁₆; or R₁₃ and R₁₄ taken together with the nitrogen towhich they are bonded represent a 4-8 membered optionally substitutedheterocyclic ring; R₁₆ for each occurrence is independently selectedfrom the group consisting of hydrogen, hydroxyl, acylamino,—N(R₁₈)COR₁₉, —N(R₁₈)C(O)OR₁₉, —N(R₁₈)SO₂(R₁₉), —CON(R₁₈)(R₁₉),—OC(O)N(R₁₈)(R₁₉), —SO₂N(R₁₈)(R₁₉), —N(R₁₈)(R₁₉), —OC(O)OR₁₈, —COOR₁₈,—C(O)N(OH)(R₁₈), —OS(O)₂OR₁₈, —S(O)₂OR₁₈, —OP(O)(OR₁₈)(OR₁₉),—N(R₁₈)P(O)(OR₁₈)(OR₁₉), and —P(O)(OR₁₈)(OR₁₉); p is 1, 2, 3, 4, 5, or6; R₁₈ for each occurrence is independently selected from the groupconsisting of hydrogen, alkyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, and heteroaralkyl; R₁₉ for each occurrence isindependently selected from the group consisting of hydrogen, alkyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, andheteroaralkyl; or R₁₈ taken together with R₁₉ represent a 4-8 memberedoptionally substituted ring; R₂₀, R₂₁, R₂₂, R₂₄, and R₂₅, for eachoccurrence are independently alkyl; R₂₃ is alkyl, —CH₂OH, —CHO, —COOR₁₈,or —CH(OR₁₈)₂; R₂₆ and R₂₇ for each occurrence are independentlyselected from the group consisting of hydrogen, alkyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl; provided thatwhen R₁ is hydroxyl, R₂ is hydrogen, R₆ and R₇ taken together form adouble bond, R₂₀ is methyl, R₂₁ is methyl, R₂₂ is methyl, R₂₃ is methyl,R₂₄ is methyl, R₂₅ is methyl, R₂₆ is hydrogen, R₂₇ is hydrogen, Q is abond, and W is oxygen; R₃ and R₄ are not both hydrogen nor when takentogether represent an unsubstituted azetidine; and the absolutestereochemistry at a stereogenic center of said formula may be R or S ora mixture thereof and the stereochemistry of a double bond may be E or Zor a mixture thereof.
 2. The pharmaceutical composition of claim 1,wherein R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, and R₂₅ are methyl; R₂₆ is hydrogen, Qis a bond; and W is oxygen.
 3. The pharmaceutical composition of claim1, wherein R₁ is hydroxyl or —OC(O)R₈.
 4. The pharmaceutical compositionof claim 1, wherein R₂ is hydrogen.
 5. The pharmaceutical composition ofclaim 1, wherein R₃ and R₄ are independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆.
 6. Thepharmaceutical composition of claim 1, wherein R₅ is hydrogen or has aformula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl.
 7. The pharmaceutical composition of claim 1,wherein R₆ and R₇ taken together form a double bond.
 8. Thepharmaceutical composition of claim 1, wherein R₂₇ is hydrogen.
 9. Thepharmaceutical composition of claim 1, wherein R₁ is hydroxyl or—OC(O)R₈; and R₂ is hydrogen.
 10. The pharmaceutical composition ofclaim 1, wherein R₁ is hydroxyl or —OC(O)R₈; R₂ is hydrogen; and R₃ andR₄ are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆.
 11. The pharmaceuticalcomposition of claim 1, wherein R₁ is hydroxyl or —OC(O)R₈; R₂ ishydrogen; R₃ and R₄ are independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, aralkyl, heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; and R₅ ishydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl.
 12. The pharmaceutical composition of claim 1,wherein R₁ is hydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ areindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl,heteroaralkyl, or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; and R₆ and R₇ taken together form a double bond. 13.The pharmaceutical composition of claim 1, wherein R₁ is hydroxyl or—OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independently hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl, or—[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ taken together form a double bond; and R₂₇is hydrogen.
 14. The pharmaceutical composition of claim 1, wherein R₁is hydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ taken together form a double bond; and R₂₇is hydrogen.
 15. The pharmaceutical composition of claim 1, wherein R₁is hydroxyl or —OC(O)R₈; R₂ is hydrogen; R₃ and R₄ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl, heteroaralkyl,or —[(CR₂)_(p)]—R₁₆; R₅ is hydrogen or has a formula 1a:

wherein R₁₇ is selected independently from the group consisting ofhydrogen, halide, hydroxyl, alkoxyl, aryloxy, acyloxy, amino,alkylamino, arylamino, acylamino, aralkylamino, nitro, acylthio,carboxamide, carboxyl, nitrile, —COR₁₈, —CO₂R₁₈, —N(R₁₈)CO₂R₁₉,—OC(O)N(R₁₈)(R₁₉), —N(R₁₈)SO₂R₁₉, —N(R₁₈)C(O)N(R₁₈)(R₁₉), and—CH₂O-heterocyclyl; R₆ and R₇ taken together form a double bond; and R₂₇is hydrogen.
 16. The pharmaceutical composition of claim 1, wherein thecompound is selected from the group consisting of:


17. The pharmaceutical composition of claim 1, wherein the compound is:


18. The pharmaceutical composition of claim 1, further comprising anantioxidant.
 19. The pharmaceutical composition of claim 1, furthercomprising a metal chelator.
 20. The pharmaceutical composition of claim1, further comprising an antioxidant and a metal chelator.
 21. Thepharmaceutical composition of claim 1, wherein said antioxidant isascorbate, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite, thioglycerol, sodium mercaptoacetate,sodium formaldehyde sulfoxylate, ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, oralpha-tocopherol.
 22. The pharmaceutical composition of claim 20,wherein said antioxidant is ascorbate.
 23. The pharmaceuticalcomposition of claim 20, wherein said metal chelator is citric acid,ethylenediamine tetraacetic acid (EDTA) and its salt, DTPA(diethylene-triamine-penta-acetic acid) and its salt, EGTA and its salt,NTA (nitriloacetic acid) and its salt, sorbitol and its salt, tartaricacid and its salt, N-hydroxy iminodiacetate and its salt,hydroxyethyl-ethylene diamine-tetraacetic acid and its salt, 1- and3-propanediamine tetra acetic acid and their salts, 1- and3-diamino-2-hydroxy propane tetra-acetic acid and their salts, sodiumgluconate, hydroxy ethane diphosphonic acid and its salt, or phosphoricacid and its salt.
 24. The pharmaceutical composition of claim 20,wherein said metal chelator is EDTA.
 25. The pharmaceutical compositionof claim 20, wherein said antioxidant is ascorbate, and said metalchelator is EDTA.
 26. A method of treating cancer, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition of claim 1, wherein the cancer isselected from the group consisting of breast cancer, multiple myeloma,Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphocytic leukemia,chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloidleukemia, malignant melanoma, lung cancer, colon cancer, ovarian cancer,and myelodysplastic syndrome.
 27. The method of claim 26, wherein themode of administration of the compound is selected from the groupconsisting of inhalation, oral, intravenous, sublingual, ocular,transdermal, rectal, vaginal, topical, intramuscular, intra-arterial,intrathecal, subcutaneous, buccal, and nasal.
 28. The method of claim26, further comprising administering to the subject a therapeuticallyeffective amount of an anti-neoplastic agent.
 29. The method of claim28, wherein the anti-neoplastic agent is selected from the groupconsisting of docetaxel, paclitaxel, imatinib mesylate, gemcitebine,bortezomib, cis-platin, carboplatin, and 5-fluorouracil.
 30. The methodof claim 28, wherein the anti-neoplastic agent is selected from thegroup consisting of docetaxel and paclitaxel.
 31. The method of claim28, wherein the anti-neoplastic agent is imatinib mesylate.
 32. Themethod of claim 26, further comprising administering to the subject atherapeutically effective amount of radiation.
 33. The method of claim28, further comprising administering to the subject a therapeuticallyeffective amount of radiation.